JP2006516535A - Immunogens and corresponding antibodies specific for high molecular weight aggregation intermediates common to amyloids formed from proteins of different sequences - Google Patents

Abstract

Compositions of matter that comprise one or more conformational epitopes found on amyloid peptide aggregates, antibodies to such epitopes and methods for making and using the compositions, eptitopes and/or antibodies. The invention includes synthetic or isolated compositions that contain or consist of certain conformational epitopes that are found on peptide aggregates (e.g., toxic peptide aggregates) present in human or veterinary patients who suffer from, or who are likely to develop, amyloid diseases (e.g., Alzheimer's Disease). The invention includes methods for the detection, treatment and prevention of diseases in humans or animals, using such compositions. The invention further includes antibodies which bind to the conformational epitopes as well as methods for making such antibodies and methods for the detection, treatment and prevention of diseases and/or identification of potential therapies (e.g., drug screening) using such antibodies.

Description

  The present invention relates generally to the fields of medicine, immunology and protein biochemistry, and more particularly to specific antigenic compositions and antibodies useful in the diagnosis, treatment and / or modeling of amyloid diseases.

  At least some of the many biological functions arise from the ability of proteins to take on various sequence-dependent structures. However, certain protein sequences can often form insoluble aggregates of unusually folded structures known as amyloid fibrils. These amyloid fibrils are thought to be involved in the pathogenesis of various amyloid diseases of genetic origin, infectious origin and / or spontaneous origin, which are spongiform encephalopathy, Alzheimer's disease, Parkinson's disease, type II diabetes, Creutzfeldt • Includes Jacob disease, Huntington's disease, possibly macular degeneration, various prion diseases and many other diseases. In at least some of these amyloid diseases, amyloid fibrils cause the development of amyloid plaques.

  Amyloid peptides are the main constituents of amyloid plaques. In the case of Alzheimer's disease, these peptides are called Aβ peptides or β-amyloid peptides. Aβ peptide is a 39-43 amino acid internal fragment of amyloid precursor protein (APP). Several mutations within the APP protein correlate with the presence of AD. For example, Goate et al., Nature (1991) 349, 704 (from valine to isoleucine); Chartier Harlan et al., Nature (1991) 353, 844 ( Murell et al., Science (1991) vol. 254, 97 (valine to phenylalanine); Mullan et al., Nature Genet. (1992) Volume 1, page 345 (double mutation changing from lysine 595-methionine 596 to asparagine 595-leucine 596). Such mutations are believed to cause AD by causing increased processing from APP to A or altered processing. In particular, processing of APP resulting in accumulation of longer forms of A, such as A1-42 and A1-43, is believed to be important in the cause of AD. Mutations in other genes (eg, presenilin genes PS1 and PS2) are thought to indirectly affect APP processing, resulting in the production of long forms of A. See, for example, Hardy, TINS (1997), Volume 20, page 154.

  European Patent Publication No. EP 526,511 (McMichael) and PCT International Patent Publication No. WO / 9927944 (Schenk) describe the administration of Aβ to patients for the treatment or prevention of Alzheimer's disease. is doing. However, although active immunization of Aβ in transgenic mice has an apparent benefit, extending this approach to AD patients resulted in undesirable inflammation of the central nervous system in some subjects. See Hardy, Dee, J. Selkoe (2002) Science Vol. 297, pages 353-356.

  Soluble Aβ includes Aβ monomers as well as aggregates of such monomers, referred to as protofibrillar aggregates. These primordial fibril aggregates result in the development of amyloid fibrils. The soluble Aβ content of the human brain correlates with the severity of AD compared to the accumulation of amyloid plaques. For example, Wai. M. YM Kuo et al. (1996) J. MoI. Biol. Chem. 271, 4077-4081; A. McLean et al. (1999) Anals of Neurology 46, 860-6; F. See LF Lue et al. (1999) American Journal of Pathology 155, 853-862. Furthermore, recent reports suggest that the toxicity of Aβ and other amyloidogenic proteins is not present in soluble monomers or accumulating insoluble fibrils, but is highly present in protofibrillar aggregates. See, for example, Hartley et al. (1999), Journal of Neuroscience Vol. 19, pages 8876-8884; Lambert et al. 1998) 95, 6448-53; and Bucciantini et al., Nature (2002) 416, 507-511; and Hartley et al. Nature (2002). See Volume 418, page 291. Taken together, these results indicate that primordial fibril aggregates may be more pathologically important than other forms of amyloid peptides and thus prevent or treat amyloid diseases such as AD Shows that it may be a more desirable target.

  There is a need for the development of antigens that can produce antibodies that bind to toxic forms of amyloid with high specificity, thereby inhibiting the pathogenicity of amyloid diseases.

  The present invention provides antigens useful for producing antibodies that specifically bind to Aβ peptide aggregates and do not bind to soluble low molecular weight Aβ or Aβ fibrils. These antibodies also specifically recognize amyloid peptide aggregates produced from all other types of amyloidogenic peptides and proteins tested herein, but the corresponding low molecular weight amyloid peptide or protein. It does not bind to fibers.

  According to the present invention, there is provided an isolated composition, eg, an antigenic composition, comprising an epitope of a fibrillar aggregate that forms in a human or animal and contributes to amyloid fibril formation, eg, a conformational epitope. The The amyloid fibrils may be free of epitopes of the composition or may be substantially free of epitopes of the composition. Further, the amyloid peptide monomer may be free of epitopes of the composition or may be substantially free of epitopes of the composition. Compositions comprising antibodies that bind to these epitopes are also provided. In one embodiment, these compositions are isolated from natural sources. In other embodiments, these compositions are synthetic. In one useful embodiment, these compositions are pharmaceutical compositions such as vaccines. Further in accordance with the present invention, these compositions comprise peptides or proteins that can be conformationally constrained. These peptides can be isolated from nature or can be synthetic. In one embodiment, the peptide consists of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and mixtures thereof. Selected from the group.

Further in accordance with the present invention, these compositions are supported by a surface that can be any shape including curved or flat. In one useful embodiment, the surface comprises a solid. The surface can be a film, particle or sheet. In one embodiment, the surface is functionally modified, eg, functionally modified to allow formation of a self-assembled peptide monolayer. Furthermore, the surface can consist of proteins, such as pleated sheets. In one embodiment, the peptide is bound to the support surface. For example, these peptides can be chemically bound to the support surface. Chemical bonds include ionic bonds, hydrogen bonds, covalent bonds and van der Waals attractions. In one particularly useful embodiment, the chemical bond is a covalent bond. These compositions can consist of linkers effective to attach these peptides to the surface. These linkers include, but are not limited to, streptavidin, hydrocarbon molecules such as citrate, HS— (CH ₂ ) _n —COOH, HS— (CH ₂ ) _n —NH ₂ , HS— (CH _{2) n -OH, HS- (CH} 2) n -COOR, phosphoramides -NH _2, includes but is disulfides -R-COOH cyclic or acidic, but not limited to hydrocarbon chains, cyclic or acidic disulfide -R- _{NH 2, Si (OCH 3)} 3-R-NH 2, Si (OCH 3) 3-R-COOH and - may include maleimides. The support may be composed of any suitable material including but not limited to gold, zinc, cadmium, tin, titanium, silver, selenium, gallium, indium, arsenic, silicon, mixtures thereof, or combinations thereof. .

  Further in accordance with the present invention, fibrillar aggregates as described herein can have a molecular weight in the range of about 10 kDa to about 100,000,000 kDa. In one embodiment, the fibrillar aggregate comprises 5 monomers. In other embodiments, the fibrillar aggregate comprises 8 monomers. This protofibrillar aggregate is present in a human or animal having a disease characterized by amyloid deposition and may include toxic species. The present invention provides antibodies that can be effective in reducing the toxicity of protofibrillar aggregates.

  Further according to the present invention, this protofibrillar aggregate is present in a human or animal having a disease characterized by amyloid deposition. For example, the disease includes Alzheimer's disease, early onset Alzheimer's disease associated with Down syndrome, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma and spongiform encephalopathy (eg, bovine spongiform encephalopathy (BSE), mad cow Disease, sheep scrapie and mink spongiform encephalopathy), Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeld-Jakob disease, Gerstmann-Streisler-Scheinker syndrome, Kool disease, lethal familial insomnia , Chronic wasting syndrome, familial amyloid polyneuropathy, frontotemporal dementia, type II diabetes, systemic amyloidosis, serum amyloidosis, British familial dementia, Danish familial dementia, macular degeneration, cerebrovascular amyloidosis, prion disease or It can be another amyloid disease.

  Further according to the present invention there is provided a method of preventing or treating a disease or condition in a human or animal subject, wherein the disease or condition is characterized by the presence of amyloid deposits. These methods can include administering to the subject a therapeutically effective or prophylactic amount of the composition. In one embodiment, the method includes inducing an immune response against the conformational epitope.

  Further according to the present invention, there is provided a method for preventing or treating a disease or condition characterized by amyloid deposition in a human or animal, wherein the method forms an antibody in the human or animal to contribute to fibril formation. Binding to a conformational epitope of the protofibrillar aggregate. In one embodiment, these methods include administering an antibody. This composition is administered by intrathecal administration, intrathecal administration, oral administration, transdermal administration, pulmonary administration, intravenous administration, subcutaneous administration, intramuscular administration, nasal administration, rectal administration, sublingual administration or buccal administration Can be done.

  Further according to the present invention there is provided a method of making an antibody that may comprise the step of administering a composition of the present invention to a human or animal. The method can also include recovering the antibody from the human or animal.

  Further according to the present invention there is provided a method of diagnosing a disease characterized by amyloid deposition, comprising a tissue or fluid from a human or animal patient, an antigenic composition of the present invention or an antibody of the present invention. And a step of mixing. In one embodiment, the tissue or fluid is cerebrospinal fluid. The disease includes, but is not limited to, Alzheimer's disease, early-onset Alzheimer's disease associated with Down's syndrome, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, and mad cow disease, sheep scrapie and mink Spongiform encephalopathy, including spongiform encephalopathy, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeld-Jakob disease, Gerstmann-Streisler-Scheinker syndrome, Kool's disease, fatal familial insomnia, chronic wasting Syndrome, transthyretin-related amyloidosis, eg familial amyloid polyneuropathy and serum amyloidosis, frontotemporal dementia, type II diabetes, systemic amyloidosis, British familial dementia, Danish familial dementia, macular degeneration and It includes vascular amyloidosis. The amount of antibody can be measured as antibody titer. In one embodiment, the amount of antibody is measured using an ELISA assay.

  Further in accordance with the present invention, there is provided a method for assessing the efficacy of a treatment method for a human or animal having a disease characterized by amyloid deposition, which method is performed in a patient-derived tissue sample prior to treatment with a drug. Determining the baseline amount of an antibody specific for the antigen comprising the composition of the present invention, and after treatment with the agent, the amount of antibody in a tissue sample from the subject is determined as the antibody baseline. A step of comparing with the amount may be included. In one embodiment, a decrease in the amount of antibody or a lack of significant difference therebetween measured after treatment and compared to the baseline amount of antibody indicates a negative treatment outcome. In other embodiments, a significantly greater amount of antibody measured after treatment and compared to the baseline amount of antibody indicates a positive treatment outcome.

  Further in accordance with the present invention, there is provided a method for assessing the efficacy of a method of treatment of a human or animal having a disease characterized by amyloid deposition, which method is performed in a patient-derived tissue sample prior to treatment with a drug. Determining the baseline amount of fibrillar aggregates specific for the antibody of the present invention and the amount of protofibrillar aggregates specific for the antibody of the present invention in a tissue sample from this subject. Comparing with a baseline amount of fibril aggregates may be included. In one embodiment, a reduction in the amount of fibrillar aggregates measured after treatment and compared to the baseline amount of fibrillar aggregates or a lack of significant difference therebetween indicates a negative treatment outcome. . In other embodiments, a significantly greater amount of fibrillar aggregates measured after treatment and compared to the baseline amount of fibrillar aggregates indicates a positive treatment outcome.

  Further according to the present invention there is provided a method of monitoring amyloid disease or susceptibility to amyloid disease in a human or animal, the method comprising detecting an immune response to the composition of the invention in a sample from a patient. obtain.

  Further according to the invention, the amount of antibody can be measured as antibody titer and the amount of antigen can be measured as antigen titer. In one embodiment, the amount of antibody is measured by an ELISA assay. In one embodiment, the amount of antigen is measured by an ELISA assay.

  Furthermore, according to the present invention, the detection of an immune response comprises the steps of detecting an antibody that specifically binds to the composition of the present invention and / or detecting a T cell that specifically reacts with the composition of the present invention. May be included.

  Further according to the present invention there is provided a diagnostic kit useful for detecting a disease characterized by amyloid deposition, the kit comprising an isolated composition of the invention comprising an antigen of the invention or an antibody of the invention. Can contain things.

  Each and every feature described herein, as well as each and every combination of two or more such features, is within the scope of the invention, unless the features contained in such combinations are inconsistent with each other. Contained within.

These and other aspects and advantages of the present invention are set forth in the following drawings, detailed description, examples, and claims.
Definitions The term “adjuvant” refers to a compound that enhances the immune response to an antigen when administered in combination with the antigen, but does not produce an immune response to the antigen when administered alone. Adjuvants can enhance the immune response by several mechanisms, including lymphocyte migration, B and / or T cell stimulation, and macrophage stimulation.

  The term “A” or “Aβ peptide” refers to peptides that make up low molecular weight soluble oligomers, fibrillar aggregates, fibrils and amyloid deposits, each associated with AD. Amyloid Aβ peptides include, but are not limited to, Aβ39, Aβ40, Aβ41, Aβ42 and Aβ43, which are 39 amino acids long, 40 amino acids long, 41 amino acids long, 42 amino acids long and 43 amino acids long, respectively. is there.

  An “amyloid peptide” is a peptide present in amyloid forms including amyloid peptide intermediates, low molecular weight soluble oligomers, amyloid fibrils and amyloid plaques.

The term “antibody” is used to include intact antibodies and binding fragments thereof, including, for example, full-length antibodies (eg, IgG antibodies) or antigen-binding moieties only (eg, Fab, F (ab ′) ₂ or scFv). Fragment), but is not limited thereto. In general, a fragment is equivalent to an intact antibody in which it has been induced for specific binding to an antigen. If desired, the antibody or binding fragment thereof can be chemically conjugated to another protein or expressed as a fusion protein with the other protein.

  An “anti-oligomer antibody” or “anti-oligomer” is an antibody that binds to an amyloid peptide aggregation intermediate, but does not bind to, or specifically bind to, a monomer, dimer, trimer or tetramer of amyloid peptide That is.

  A composition or method “comprising” one or more of the listed elements may include other elements not specifically recited. For example, a composition comprising amyloid Aβ peptide can include isolated amyloid Aβ peptide as both a component of a larger polypeptide sequence or as part of a composition comprising multiple elements.

  The term “epitope” or “antigenic determinant” refers to the site on an antigen to which B and / or T cells respond, or the site on the molecule from which the antibody is produced and / or the site to which the antibody binds. That is. For example, an epitope can be recognized by an antibody that defines it.

  A “linear epitope” is an epitope whose primary sequence constitutes the recognized epitope. Linear epitopes generally comprise at least 3 amino acids, more usually at least 5 amino acids, such as from about 8 to about 10 amino acids, in a unique sequence.

  A “conformational epitope” is, in contrast to a linear epitope, an epitope in which the primary sequence of amino acids that make up the epitope is not a single component that defines the recognized epitope (eg, the primary sequence of amino acids Epitopes that are not necessarily recognized by the antibody defining the epitope). In general, a conformational epitope consists of an increased number of amino acids compared to a linear epitope. With respect to recognition of conformational epitopes, this antibody recognizes the three-dimensional structure of the peptide or protein. For example, when a protein molecule is folded to form a three-dimensional structure, the specific amino acid and / or polypeptide backbone that forms the conformational epitope is juxtaposed to allow the antibody to recognize the epitope. Methods for determining epitope conformation include, but are not limited to, x-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy and site-specific spin labeling and electron paramagnetic resonance spectroscopy. For example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glen E. et al. See Glenn E. Morris, Ed. (1996), the disclosure of which is hereby incorporated by reference in its entirety.

The term “immunological response” or “immune response” refers to a beneficial humoral (antibody-mediated) response to amyloid peptides and / or cellular (antigen-specific T cells or their secreted products) in a recipient patient. Related to the generation of responses). Such a response can be an active response induced by administration of an immunogen or a passive response induced by administration of antibody or primed T cells. The cellular immune response is guided by the presentation of polypeptide epitopes associated with class I or class II MHC molecules and activates antigen-specific CD4 ⁺ T helper cells and / or CD8 ⁺ cytotoxic T cells. This response may also include monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils or other components of innate immunity.

An “immunogenic factor”, “immunogen” or “antigen” can be combined with an adjuvant, if necessary, to induce an immunological response against itself upon administration to a subject.
“Isolated” means purified, substantially purified, or partially purified. Isolated may also mean present in an environment other than the natural environment. For example, an antibody that is not present in the whole serum in which the antibody is naturally found when it occurs in nature is an isolated antibody.

  “Low molecular weight aggregates”, “low molecular weight amyloid aggregates”, “low molecular weight oligomers” and “low molecular weight soluble oligomers” are amyloid peptides in which the peptide is present in less than 4 or 5 aggregates. is there. In one particular example, low molecular weight Aβ is a low molecular weight soluble oligomer found in association with AD.

  The term “patient” includes human and other animal subjects undergoing therapeutic, prophylactic or diagnostic treatment, or a human or animal having or susceptible to a disease.

  “Primitive fibril aggregate”, “micelle aggregate”, “high molecular weight aggregated intermediate”, “high molecular weight amyloid peptide aggregate”, “high molecular weight soluble amyloid peptide aggregate”, “amyloid peptide aggregate”, “soluble” An “aggregation intermediate”, “amyloid oligomer intermediate”, “oligomer intermediate” and “oligomer aggregate”, or simply “intermediate” is more than three individual peptide monomers or protein monomers, for example more than four An aggregate containing peptide monomers or protein monomers. Upper size primordial fibrillar aggregates comprise oligomeric aggregates, which form globular structures or micelles and micelle spines that lead to fibril formation.

  “Cyclic primordial fibrils” are primitives in which 3-10 spherical oligomer subunits are arranged in a circular or circular fashion with cavities appearing as pores in an electron micrograph or atomic force micrograph. It is a specific subunit of fiber aggregates.

  The molecular weight of the primordial fibril aggregate can be in the range of about 10 kDa to about 100,000,000 KDa, such as about 10 kDa to about 10,000,000 KDa or 1,000,000 KDa. However, this size range is not limited, and protofibrillar aggregates are not defined by the molecular weight range.

“Primordial fibrils” are primordial fibrillar aggregates comprising globular structures containing amyloid Aβ peptides that appear to show spines of globular structures that form a curvilinear structure.
“Specific binding” between two entities means an affinity of at least 10 ⁶ M ⁻¹ , 10 ⁷ M ⁻¹ , 10 ⁸ M ⁻¹ , 10 ⁹ M ⁻¹ , or 10 ¹⁰ M ⁻¹ . Affinities higher than 10 ⁸ M ⁻¹ are preferred for specific binding.

  The term “substantially identical” means, for example, that two peptide sequences are at least 65% aligned if necessary by the program GAP or BESTFIT with default gap weights. Share at least 80% or 90% sequence identity, or at least 95% or more sequence identity, such as 99% or more sequence identity.

  Preferably, residue positions in non-identical alignments differ in conservative amino acid substitutions, ie substitutions of other amino acids with the same class or group of other amino acids. Some amino acids can be classified as follows: Group I (hydrophobic side chains): leu, met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; group III (acidic side chain): asp, glu; group IV (basic side chain): asn, gln, his, lys, arg; group V (residues affecting chain orientation): gly, pro And Group VI (aromatic side chain): trp, tyr, phe. Non-conservative substitutions may involve exchanging one member of these classes with a member of another class.

  For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, sequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. This sequence comparison algorithm can then be used to calculate% sequence identity for the test sequence relative to the reference sequence, based on the designated program parameters. The optimal alignment of sequences for comparison is described, for example, in Smith and Waterman, Adv. Appl. Math. Volume 2: Page 482 (1981), Local Homology Algorithm, Needleman and Wunsch, J. Am. Mol. Biol. 48: 443 (1970), Homology Alignment Algorithm, Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988) search methods for similarity, these algorithms (GAP, BESTFIT, FASTA and TFSTA in Wisconsin Genetics Software Package, Genetics Computer Group, Wisconsin, Wisconsin) , Science Drive 575 (575 Science Dr., Madison), computer-implemented, or by visual inspection.

  One example of an algorithm that is suitable for determining% sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). In general, default program parameters can be used to perform sequence comparisons, but customized parameters can also be used. For amino acid sequences, the BLASTP program uses by default a word length of 3 (W), an expected value of 10 (E) and a BLOSUM62 scoring matrix. See, for example, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89, 10915 (1989). Conservative substitutions include substitutions between amino acids in the same class.

“Synthetic” means not naturally occurring. For example, a synthetic composition is a composition that is wholly or partly not found in nature.
A “therapeutic agent” or “therapeutic agent” is a substance useful for the treatment or prevention of a disease in a patient. The therapeutic agents of the present invention are generally substantially pure. This is that the drug is generally at least about 50 w / w (weight / weight) pure and substantially free of proteins and contaminants that interfere with the efficacy of the therapeutic agent. These agents may be at least about 80% w / w, more preferably at least 90% w / w or about 95% w / w purity. However, using conventional protein purification techniques, 99% w / w or more of the same peptide can be produced.

  The present invention relates to compositions having one or more epitopes found on amyloid peptide aggregates present in humans or animals having a disease characterized by amyloid deposition, antibodies to such epitopes, and compositions thereof And methods for making and using the antibodies are provided.

  More particularly, the present invention includes, but is not limited to, epitopes (eg, conformational epitopes) found on peptide aggregates (eg, amyloid peptide aggregates) in humans or animals having a disease characterized by amyloid deposition. ), Methods of making these compositions, methods of using these compositions (including but not limited to those for disease detection, treatment and prevention), these compositions Antibodies to conformational epitopes present on objects, methods of making these antibodies and methods of using these antibodies, including but not limited to those for disease detection, treatment and prevention Is included.

  Amyloid disease is characterized by a predisposition to amyloid plaques or precursor accumulation in amyloid plaques in a patient, or accumulation of precursors to amyloid plaques or amyloid plaques in a patient. One of the main components of amyloid plaques is amyloid peptide. Although the general conformation of amyloid peptides can vary from disease to disease, the peptides often have a characteristic-pleated sheet structure. Amyloid peptides include peptides and proteins that are about 10 amino acids long or about 20 amino acids long to about 200 amino acids long. This size range is not limited and the present invention contemplates amyloid peptides or proteins having fewer or more amino acids.

  Primitive fibril aggregates are intermediates in the production of insoluble fibrils that accumulate in amyloid plaques of humans or animals with diseases characterized by amyloid deposition (eg, Alzheimer's disease). Primordial fibril aggregates are as small as 4 amyloid peptides, as small as 5 amyloid peptides, as small as 6 amyloid peptides, as small as 7 amyloid peptides, or 8 Including aggregates that can be as small as the amyloid peptide of. In one embodiment, the primordial fibril aggregate is a micellar aggregate or micelle or micelle spine. Primordial fibril aggregates are effective in forming conformational epitopes recognized by the antibodies of the present invention.

  The conformational epitopes of the present invention found on primordial fibril aggregates are also present in their characteristic cross-βx rays in the unique precursor proteins of amyloid peptides (eg, monomers, dimers, trimers or tetramers of amyloid peptides). It is virtually absent in mature amyloid fibrils and amyloid plaques as defined by the fiber diffraction pattern. Primordial fibril aggregates comprising specific polypeptide structures that produce conformational epitopes recognized by the antibodies of the present invention are approximately pentamer, hexamer, heptamer or octamer to 1,000,000. Has a micellar form with a molecular weight greater than daltons or a size range of protofibrils. The immunogen of the present invention includes compositions comprising these epitopes. The antibodies of the present invention are effective in binding to these epitopes.

  The immunogens of the present invention can be obtained from any suitable source. For example, these immunogens can be purified from naturally occurring sources. In one particularly useful embodiment, these immunogens are synthetic.

Immunogens presenting the epitopes necessary to produce the antibodies of the invention can be prepared as oligomeric intermediate mimetics comprising amyloid peptides or proteins. For example, Aβ peptides (eg A40 and A41), -synuclein, IAPP (C2A and C7A, where alanine is replaced by a naturally occurring cysteine in IAPP), polyglutamine KKQ40KK or polyglutamine (where The number of Q residues is greater than 32), calcitonin, TTR and its variants TTR Pro ⁵⁵ , TTR Phe ⁷⁸ , vitronectin, polylysine, polyarginine, serum amyloid A, cystantin C, IgGκ light chain Other amyloid peptides disclosed herein and amyloid peptides associated with each amyloid disease disclosed herein may be used.

  Peptides useful in the present invention can be obtained from natural sources, eg, can be purified or produced from naturally occurring sources. Manufacturing methods include any suitable method, including but not limited to solid phase synthesis and heterologous gene expression.

  The fact that the antigens of the present invention are common to widely varying primary sequences of amyloid indicates that this epitope is formed from a specific three-dimensional conformational polypeptide backbone referred to as a conformational epitope.

  Solid phase synthesis and purification of peptides is described in D.C. Continuous flow semi-automatic as described in D. Burdick et al. (1992) J Biol Chem Vol. 267, 546-54, the disclosure of which is incorporated herein by reference. It can be performed by fluoren-9-ylmethoxycarbonyl chemistry using an instrument.

  Briefly, the first Fmoc-amino acid is manually coupled to sulfurylbutyry-AM-PEGA resin (Novabiochem, San Diego, Calif.) In dichloromethane (DCM). Diisopropylethylamine (DIEA) was added, the mixture was stirred at room temperature for 20 minutes, cooled to -10 ° C to -20 ° C, and ByBop (benzotriazol-1-yl-oxy-tris (pyrrolidino)) -Phosphonium hexafluorophosphate) is added. The mixture is stirred at -10 ° C to -20 ° C for 8-9 hours. Coupling efficiency can be checked using the Kaiser test, which is well known in the field of peptide synthesis.

  Acetylation can be performed using acetic anhydride. Amino acid chain elongation is by fluoren-9-ylmethoxycarbonyl chemistry using a continuous flow semi-automatic instrument. This peptide was synthesized in a synthesis vessel with N-methyl-2-pyrrolidone 5 × (NMP) 5.0 mL NMP, 185 μL i-Pr 2 EtN (1.1 mmol) and 400 μL iodoacetonitrile (previously in the dark with an alumina basic filter). Filtered on the bed). The reaction mixture is then stirred for 24 hours on a rotary plate and in the dark. The resin is washed with 5 × NMP and 5 × DMF, then washed using 5 × CH 2 Cl 2 and then dried. The resin is washed with 5 × THF and then THF and TMS—CH 2 N 2 (50:50, v / v, hexane) are added. After stirring for 2 hours, the resin is washed with THF and DMF.

  This resin is added to 120 μL of ethyl-3-mercaptopropionate and the mixture is stirred on a rotary plate for 24 hours. The resin is filtered and then washed with 3 × 3 ml DMF. The filtrate and washings are collected and rotary evaporated at 34 ° C.

  The resulting peptide is deprotected using standard methods (TFA and scavenger) and purified by RP-HPLC. Its purity can be checked by analytical RP-HPLC and electrospray mass spectrometry.

  These peptides can also be produced by standard heterologous gene expression methods. For example, the recombinant expression can be in bacteria (eg, E. coli) or yeast, insect cells or mammalian cells. Procedures for recombinant expression are described in Sambrook et al., Molecular Cloning: A Laboratory Manual (C.S.H.P. Press), NY Location, 2nd edition, 1989). In addition, a number of amyloid peptides, including human insulin or lysozyme, are available from commercial sources.

  Peptides useful in the present invention can be advantageously aggregated or conformationally constrained to form useful epitopes as described herein. In one useful embodiment, these peptides are bound to the surface and physically attached to the surface in a manner that allows, for example, production of epitopes recognized by the antibodies of the invention, or Chemically bonded.

  For example, the C-terminal thioester can be attached to these peptides in a conventional manner as is known to those skilled in the art. For example, the C-terminal thioesterification of these peptides by Fmoc chemistry is substantially similar to Inginito, R. et al. (1999) Journal of the American Chemical Society Vol. 121, pp. 11369-11374. Can be implemented as described in. C-terminal thioesterified peptides are readily attached to surfaces such as metal surfaces.

  The surface to which these peptides are bound or attached can be any suitable surface. For example, the surface can be solid. The surface can include one or more of hydrocarbons, polymer (s), plastic, glass, metal, ceramic or one or more biomolecules (eg, proteins, fats, nucleic acids and carbohydrates). One or more of these components can constitute this surface. For example, the particles can consist of a polymer coated with a metal. This surface can be flat or have a three-dimensional shape, such as a curved surface. Further, the surface can be a particle. In one embodiment, oligomeric aggregate molecular mimics are produced using nanospheres. These nanospheres can be of any suitable size. For example, the diameter of these nanospheres can be in the range of about 0.01 nm to about 1 cm. In one useful embodiment, the diameter of these nanospheres is about 5 nm.

  In one particularly useful embodiment, gold nanospheres are used to produce molecular mimetics. Briefly, these nanospheres can be incubated for 3 hours in a 0.2 mg / ml C-terminal thioester peptide (pH 5.0-5.5) solution followed by 100 mM Tris pH 8.0 (0.0. The pH is adjusted to 7.4 using 2% sodium azide). After a 6 hour incubation at room temperature, these molecular mimetics were recovered by centrifugation, washed 3 times with PBS pH 7.6 to remove unincorporated peptide, and then 0.02% azide Stored in sodium at 4 ° C. A collection of such molecular mimetics is shown in FIG. However, this is an example of a method for producing the molecular mimics of the present invention. Other methods of producing these mimetics will be readily apparent to those skilled in the art.

  Although the present invention recognizes epitopes present on amyloid intermediates, epitopes present on amyloid monomers, dimers, trimers or tetramers, as well as mature amyloid fibril epitopes, aggregate amyloid peptides aggregated into insoluble masses. Antibodies that also do not recognize the epitope of the amyloid deposit that they contain are included.

  The antibodies of the present invention can be made by any suitable means. For example, these antibodies can be produced in laboratory animals. In one such case, New Zealand white rabbits, Balb / C, C57 / Black6 mice or domestic dogs are injected with a large amount of molecular mimic produced as described above. Antigen is mixed with alum adjuvant, Freund's incomplete adjuvant prior to injection, or no adjuvant (PBS only). For the first injection, the same amount of antigen and adjuvant are used. In subsequent injections, the antigen is mixed with an adjuvant and each is injected, for example, at 2-week intervals. Animals can be injected subcutaneously in small increments of 0.1 mL per checkerboard style site on the shoulder.

  This antigen is constrained in a conformation, resulting in the production of a conformation-dependent epitope antigen recognized by the antibody and presentation of this antigen on a solvent accessible surface. Specifically, attachment of the carboxyl terminus to the surface substrate maintains this region in close juxtaposition to the surface, which mimics the sequence of the Aβ peptide in the soluble oligomer [Garson-Rodriguez ), 2000 # 6890]. The attachment of the carboxyl terminus to the solid support prevents rearrangement of this region of the peptide that occurs during the structural transition from soluble oligomers to amyloid fibrils. In amyloid fibrils, this carboxyl terminus is freely mobile and is found on the solvent accessible surface of amyloid fibrils [Garson-Rodriguez, 2000 # 6890] [Torok, 2002 # 8927] [Antzutkin, 2003 # 10555]. Attachment of the carboxyl terminus to the solid support also maintains a parallel arrangement of the polypeptide chains and dissociates the polypeptide into its monomeric form, or low molecular weight monomeric, dimeric, trimeric and tetrameric forms. To prevent.

  Preferred antibodies of the invention appear to retain binding for more than 1 hour wash time and thus exhibit a relatively high binding affinity. In at least some of the antibodies of the invention, the half-life for dissociation is greater than 1 hour.

  This antibody recognizes an epitope regardless of sequence, and this epitope is shared or shared by a wide range of soluble oligomers derived from amyloidogenic peptides and proteins. This epitope is absent or has a substantially reduced structure or accessibility in the low molecular weight forms of these peptides and in amyloid fibrils. This epitope is a common structural and conformational feature of this peptide, which includes a specific conformation of the polypeptide backbone formed by a number of different protein and peptide sequences. Is not limited to). This epitope is recognized by the antibody, so that binding of the antibody to the epitope substantially reduces or eliminates the toxicity of the soluble oligomer, regardless of the protein or peptide sequence presenting the epitope. However, this description of the present invention is specific for different sequences, ie, such antibodies are possible, and these antibodies bind to the same epitope on one peptide, but these antibodies It should be understood that antibodies that may be specific and do not recognize all other amyloids are not necessarily excluded.

  For serum recovery, the IgG fraction can be affinity purified and eluted, eg, on protein G-Sepharose beads, and then dialyzed against PBS. An intermediate aggregate-specific antibody is produced on this molecular mimic by mixing the amyloid oligomer intermediate molecular mimic produced as described above with the IgG fraction, incubating for about 2 hours, and then washing. Can be purified by adsorption. After elution, the antibody can be dialyzed against PBS and stored at 4 ° C. or −70 ° C. in PBS containing 0.02% sodium azide.

  Polyclonal sera produced by vaccination of rabbits, dogs or other animals with the molecular mimetics disclosed herein are specific for amyloid peptide aggregation intermediates and are soluble low molecular weight amyloid Species or fibrillar amyloid species is not detectably reactive. See Example 4. Surprisingly, anti-oligomer immunoreactivity to low molecular weight aggregates or fibrils is not observed for unfractionated sera, indicating that the immune response to this molecular mimetic is very specific. Show. For example, antibodies raised against Aβ molecular mimetics do not bind to Aβ low molecular weight aggregates or Aβ fibrils, even after boosting rabbits with Aβ molecular mimics for 12 hours.

  Antibodies raised against Aβ peptide aggregate mimetics have been shown to bind to all other amyloid-type amyloid aggregation intermediates tested. See FIG. Furthermore, these antibodies have been shown to neutralize the toxicity of oligomeric forms of all toxic amyloids tested (ie, amyloid intermediates). See FIGS. 7, 8 and 9. It is suggested that amyloid intermediates share a common structure. Accordingly, the present invention provides that antibodies produced using molecular mimetics comprising one type of amyloid peptide are antibodies (eg, steric) that are specific for other amyloid peptide intermediate types (eg, all amyloid peptide intermediate types). Conformation dependent antibodies) are intended to be produced. For example, an antibody prepared from a molecular mimic comprising a synuclein peptide not only specifically reacts with synuclein fibrillar aggregates, but also other amyloid oligomeric intermediate forms (eg, all other amyloid oligomeric intermediates). It reacts specifically with oligomer intermediates in the form of body.

Each of the following amyloid peptides has been shown to form amyloid peptide aggregates that produce conformational epitopes recognized by antibodies of the invention (eg, antibodies raised against Aβ peptide oligomer intermediates). ing. Some of these peptides are present during amyloid deposition in humans or animals with diseases characterized by amyloid deposition. The present invention is not limited to the listed peptide or protein sequences or to specific diseases associated with some of these sequences. The present invention contemplates antibodies as described herein that bind to other amyloid peptide aggregates or all other amyloid peptide aggregates. In particular, the present invention contemplates the application of the methods and compositions of the present invention to other peptide or protein sequences that form amyloid precursor aggregates associated with other diseases.
A40 (SEQ ID NO: 1)
DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV
A42 (SEQ ID NO: 2)
DAEFRHDSGY EVHHQKLVFF AEDVGSNNKGA IIGLMVGGVV IA
Human IAPP (SEQ ID NO: 3)
KCNTATCATQ RLANFLVHSS NNFGAILSST NVGSNTY
Human prion 106-126 (SEQ ID NO: 4)
KTNMKHMAGA AAAGAVVGGL G
Stephani and co-workers (Bucciantini et al (2002) Nature 416, 507-511) have recently produced amyloid peptide aggregates formed from non-disease-related proteins. Have been reported to be cytotoxic, suggesting that these amyloid peptide aggregates may have a structure in common with disease-related amyloid peptides. Non-disease related amyloid peptide aggregates, including the following non-disease related amyloid peptides, have also been shown to bind to the antibodies of the invention.
Polyglutamine synthetic peptide KK (Q40) KK (SEQ ID NO: 5)
KKQQQQQQQQ QQQQQQQQQQ QQQQQQQQQQ QQQQQQQQQQ QQKK
Human lysozyme (SEQ ID NO: 6)
MKALIVLGLV LLSVTVQGKV FERCELARTL KRLGMDGYRG SLANWMCLAK WESGYNTRAT NYNAGDRSTGD
Human insulin (SEQ ID NO: 7)
MALWMRLPL LALLALLWGPD PAAAFVNQHL CGSHLVELY LVCGGERGFFY TPKTRREAED LQVGQVELGGG PGAGSLQPL ALEGSSLQKRG IVEQCCCTSIC SLYQLENYCN
Human transthyretin (SEQ ID NO: 8)
MASHRLLLLC LAGLVFVSEA GPTGGESKC PLMVKVLDAV RGSPAINVAV HVFRKAADDT WEPFASGKTS ESGELHGSTV
Human α-synuclein (SEQ ID NO: 9)
MDVFGMGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVEGMPQQVTGVEGDGQ
In addition, variants and fragments of wild type A42, A40 (including but not limited to A42 (A21G) Flemish mutation, A42 (E22Q) Dutch mutation, A42 (E22G) Arctic mutation, A42 (D23N) lower ( lowa) type mutation, A40 (A21G) Flemish type mutation, A40 (E22Q) Dutch type mutation, A40 (E22G) Arctic type mutation, A40 (D23N) Lower type mutation, A40 (E22Q & D23N) Dutch type & Lower type mutation, Aβ3- 42 (pGlu3), Aβ3-40 (pGlu3), A8-42, A17-42, A1-16, A3-11, A25-35, A4-16 (three analogs: Cys ¹⁶ A4-16, Ala ⁴ A4-16 and Ala ¹⁰ A4-16), His6 A40C40 (amino terminus of AβC40) The oligomeric intermediate formed from 6) is recognized by the antibody of the present invention. Other oligomeric intermediates recognized by the antibodies of the present invention include, but are not limited to, IAPP (C2A and C7A) (where alanine is replaced by a naturally occurring cysteine in IAPP), Polyglutamine KKQ40KK or polyglutamine (where the number of Q residues is greater than 32), calcitonin, TTR and its variants TTR Pro ⁵⁵ , TTR Phe ⁷⁸ , vitronectin, polylysine, polyarginine, serum amyloid A, cystatin C, Oligomer intermediates formed from IgG kappa light chains, oligomeric intermediates produced from other amyloid peptides disclosed herein, and amyloid intermediates associated with each amyloid disease disclosed herein Is included.

  The present invention provides a therapeutic agent for amyloid diseases that induces a specific immune response against amyloid oligomer intermediates. These therapeutic agents include a conformational epitope of an aggregate of amyloid peptide, a conformational epitope of an aggregate of a variant of this amyloid peptide, and / or an antibody of the present invention. Molecular mimetics containing conformational epitopes of aggregates of analogs and mimetics of amyloid peptides that cross-react with and antibodies or T cells reactive with such antibodies. Induction of an immune response is active when the immunogen is administered to induce antibodies or T cells that are specifically reactive with the amyloid peptide intermediate in the patient, or to the amyloid peptide intermediate in the patient. It may be passive when an antibody that specifically binds itself is administered.

  Analogs include allelic variants, species variants and derived variants. Analogs generally differ from naturally occurring peptides at one or several positions, often by virtue of conservative substitutions. Analogs generally exhibit at least 80% or 90% sequence identity with the natural peptide. Some analogs also include unnatural amino acids or N-terminal or C-terminal amino acid modifications. Examples of non-natural amino acids are A-2 substituted amino acids, -alkyl amino acids, lactic acid, 4-hydroxyproline, carboxyglutamic acid, eN, N, N-trimethyllysine, eN-acetyllysine, O-phosphoserine ( O-phospgoserine), N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, and wN-methylarginine.

  Therapeutic agents also include aggregates of longer peptides or proteins, including, for example, amyloid peptides, active fragments or analogs along with other amino acids. For example, the Aβ peptide can exist as an intact APP protein or a segment thereof (eg, a C-100 fragment starting at the N-terminus of Aβ and continuing to the end of APP). Such polypeptides can be screened for prophylactic or therapeutic efficacy in animal models, as described below. Aβ peptides, analogs, active fragments or other polypeptides can be administered in a form that provides an immune response against low molecular weight oligomers or three-dimensional or conformational epitopes substantially absent on fibrils.

  Therapeutic agents also include peptides and other compounds, which do not necessarily have significant amino acid sequence similarity to the amyloid protein, but nevertheless an immune response to the conformational epitope of the amyloid oligomeric intermediate. Function as an antigen of the present invention. For example, any peptides and proteins that form a pleated sheet can be screened for suitability. Anti-idiotypic antibodies against monoclonal antibodies against amyloid intermediates or synthetic mimetics of amyloid intermediates can also be used. Such an anti-Id antibody mimics an antigen and produces an immune response against the antibody (Essential Immunology, edited by Roit, Blackwell Scientific Publications, Palo Alto) 6th edition), page 181).

  Random libraries of peptides or other compounds can also be screened for suitability for use herein. Combinatorial libraries can be generated for many types of compounds that can be synthesized in a step-wise fashion. Such compounds include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomer-substituted glycines and oligocarbamates. It is. A large combinatorial library of these compounds is available from Affymax, WO 95/12608, Affimax, WO 93/06121, Columbia University, WO 94/08051, Pharmacopeia, WO 95/35503, and Scripts. (Scripts), WO 95/30642, each of which is incorporated by reference for all purposes, may be performed by the encoded synthetic libraries (ESL) method. Peptide libraries can also be generated by phage display methods. See, for example, Devlin, WO 91/18980.

  Any peptide aggregate, natural or synthetic, or other compound of interest binds to an antibody or lymphocyte (B or T) known to be specific for the oligomeric intermediate By determining their ability to do so, they can be initially screened for suitability as immunogens or antigens for use herein. For example, an initial screen can be performed with any polyclonal serum or monoclonal antibody against the oligomeric intermediate.

  Compounds identified by such screening can be further analyzed for the ability to induce antibodies or reactive lymphocytes against oligomeric intermediates. For example, multiple dilutions of serum are tested on microtiter plates pre-coated with oligomeric intermediate mimetics or purified oligomeric intermediates of the invention, and standard ELISAs are tested for reactive antibodies. Can be implemented. The compounds can then be tested for prophylaxis and therapeutic efficacy, as understood in the art, for example, in transgenic animals susceptible to amyloidogenic disease. Such animals include, for example, mice carrying the APP 717 mutation described by Games et al., Supra, as well as McConlogue et al., US Pat. No. 5,612. 486 and Hsiao et al. (1996) Science 274, 99; Staufenbier et al., Proc. Natl. Acad. Sci. USA (1997) 94, 13287-13292; Churchiller-Pierrat et al., Proc. Natl. Acad. Sci. USA, 94, 13287-13292; mice carrying the Swiss-type mutation of APP as described by Borchelt et al., Neuron (1997) 19, 939-945. The same screening approach can be used for other potential therapeutic agents, including those described above.

  The therapeutic agents of the invention also include antibodies that specifically bind to oligomeric intermediates. Such antibodies can be monoclonal or polyclonal. In one useful embodiment, these antibodies bind to a conformational epitope. Production of non-human (eg, mouse or rat) monoclonal antibodies can be accomplished, for example, by immunizing the animal with an oligomeric intermediate mimic of the invention. It is also contemplated to immunize animals with purified amyloid intermediates.

  Humanized forms of the murine antibodies of the invention can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. Queen et al., Proc. Natl. Acad. Sci. USA 86, 1000029-10033 (1989) and WO 90/07861, incorporated by reference for all purposes.

  Human antibodies can be obtained using phage display methods. See, for example, Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047. In these methods, a library of phage is generated in which members display different antibodies on their outer surface. Phages displaying antibodies with the desired specificity are selected by affinity enrichment. Human antibodies against oligomeric intermediates can also be produced from non-human transgenic mammals having a transgene encoding at least a segment of a human immunoglobulin locus and an inactivated endogenous immunoglobulin locus. For example, Lonberg et al., WO 93/12227 (1993); Kucherlapati, WO 91/10741 (1991), each of which is incorporated by reference in its entirety for all purposes. )checking. Human antibodies having the same epitope specificity as a particular mouse antibody can be selected by competitive binding experiments or other methods. Such antibodies are particularly likely to share the useful functional properties of mouse antibodies.

  Human polyclonal antibodies can also be provided in the form of serum from humans immunized with the immunogens of the invention. If desired, such polyclonal antibodies can be concentrated by affinity purification, for example using the immunogen of the invention as an affinity reagent.

Human or humanized antibodies can be designed to have IgG, IgD, IgA and IgE constant regions and any isotype (including IgG1, IgG2, IgG3 and IgG4). Antibodies are tetramers comprising two light chains and two heavy chains, as separate heavy chains, light chains, as Fab, Fab′F (ab ′) ₂ and Fv, or with variable domains of heavy and light chains. It can be expressed as a single chain antibody linked via a spacer.

  Therapeutic agents for use in the methods of the invention can also include T cells that bind to amyloid oligomeric intermediates. For example, T cells are activated for intermediates by expressing human MHC class I and human-2-microglobulin genes from insect cell lines, whereby empty complexes are formed on the surface of these cells. Formed on and capable of binding to an oligomeric intermediate antigen. T cells in contact with this cell line can be specifically activated for this antigen. See Peterson et al., US Pat. No. 5,314,813. Insect cell lines expressing MHC class II antigens can also be used to activate CD4 T cells.

  In certain instances, it may be desirable to link an immunogen of the present invention to a suitable carrier. Suitable carriers include serum albumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxin or toxoids from other pathogenic bacteria (eg, diphtheria, E. coli, cholera or H. poriori) Or an attenuated toxin derivative. Other carriers for stimulating or enhancing the immune response include cytokines (eg, IL-1, IL-1 and IL-1β peptides, IL-2, INF, IL-10, GM-CSF and chemokines (eg, , M1P1 and M1P1β, and RANTES)). The immunogen can also be linked to a peptide that enhances transport across tissues, as described in O'Mahoney's WO 97/17613 and WO 97/17614.

  The immunogens of the invention can be linked to a carrier by chemical cross-linking. Techniques for linking the immunogen to the carrier include N-succinimidyl-3- (2-pyridyl-thio) propionate (SPDP) and succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). Used disulfide bond formation (if the peptide lacks a sulfhydryl group, this may be provided by the addition of a cysteine residue). These reagents produce disulfide bonds between themselves and the cysteine residues of the peptide on one protein and amide via e-amino on lysine or other free amino groups in other amino acids. Create a bond. A variety of such disulfide / amide forming agents are described in Immun. Rev. 62, page 185 (1982). Other bifunctional coupling agents form thioethers rather than disulfide bonds. Many of these thioether formers are commercially available and include the reaction of 6-maleimidocaproic acid, 2-bromoacetic acid and 2-iodoacetic acid, 4- (N-maleimido-methyl) cyclohexane-1-carboxylic acid. Sex esters are included. These carboxyl groups can be activated by combining these carboxyl groups with succinimide or 1-hydroxy-2-nitro-4-sulfonic acid sodium salt.

  Peptides contained in the immunogens of the present invention can also be expressed as fusion proteins. The peptide can be linked to a carrier at the amino terminus, carboxyl terminus, or internally. For example, these peptides can be fused to a carrier or any useful peptide or protein sequence.

  Patients receiving treatment include individuals at risk for disease but not showing symptoms, as well as patients currently showing symptoms. In the case of certain amyloid diseases, including AD, virtually everyone is at risk of getting the disease.

  Thus, the compositions of the invention can be administered prophylactically to the general population, for example, by vaccines, without assessing the subject patient's risk. The methods of the present invention are particularly useful for individuals with a known genetic risk of amyloid disease (eg AD). Such individuals include individuals with relatives who have experienced amyloid disease, and individuals whose risk has been determined by analysis of genetic or biochemical markers, or the onset or actual presence of such diseases. Individuals who exhibit symptoms or prodromal symptoms may be included. For example, genetic markers of risk for AD include mutations in the APP gene, particularly mutations at positions 717 and 670 and 671 (referred to as Hardy mutation and Swiss mutation, respectively) (Hardy). ), TINS (see above)). Other markers of risk for AD are mutations in the presenilin genes (PS1 and PS2) and ApoE4, a family history of AD, hypercholesterolemia or atherosclerosis.

  Symptoms of amyloid disease will be apparent to a physician of ordinary skill. For example, individuals currently suffering from Alzheimer's disease can be recognized from characteristic dementia, as well as the presence of the risk factors described above. In addition, a number of diagnostic tests are available to identify individuals with amyloid disease. For example, in the case of AD, these include measurements of CSFτ level and A42 level. Elevated τ levels and decreased A42 levels indicate the presence of AD.

  In asymptomatic patients, treatment can begin at any age (eg, 10, 20, 30, 40, 50, 60, or 70 years). Treatment can result in one or more doses (eg, multiple doses over a period of time). Treatment is by analyzing the response of antibodies or activated T cells or B cells over time to this therapeutic agent (eg, oligomeric intermediate mimic) or by measuring the level of protofibrillar aggregates present over time. Can be monitored by analyzing manually. In one embodiment, treatment by administration of a single therapeutic agent of the invention (eg, a single immunogen of the invention) is as a treatment for two or more amyloid diseases (eg, all amyloid diseases). Or may serve as a prophylactic measure against these amyloid diseases.

  In prophylactic applications, the composition or medicament of the present invention eliminates or reduces the risk of this disease in patients susceptible to or at risk for this particular disease, or It is administered in an amount sufficient to delay the onset of the disease. In therapeutic applications, the composition or medians may cure or at least partially cure the symptoms of the disease and its complications to patients suspected of such diseases or patients already suffering from such diseases. Is administered in an amount sufficient to stop it. An amount adequate to accomplish this is defined as a therapeutically effective amount or a pharmaceutically effective amount. In both prophylactic and therapeutic regimens, the therapeutic agent is usually administered in several dosage forms until a sufficient immune response is achieved. Generally, this immune response is monitored and repeated dosages are given when the immune response begins to decay.

  An effective amount of the composition of the present invention will vary in the number of different factors (treatment means, target site, patient physiological state, whether the patient is a human or animal, other Medication, and whether treatment is for prophylactic or therapeutic purposes). Usually this patient is a human, but in some diseases (eg, mad cow disease), this patient is a non-human mammal (eg, a bovine subfamily), or in the case of Alzheimer's disease, The patient can be a dog. The therapeutic dosage needs to be titrated to optimize safety and efficacy. The amount of immunogen depends on whether an adjuvant is also administered, and higher dosages are required in the absence of adjuvant. The amount of immunogen for administration often varies from about 1 μg to about 500 μg per patient, and more usually from about 5 μg to 500 μg per injection for human administration. Sometimes higher doses of about 1 mg to about 2 mg per injection are used. Typically about 10 μg, about 20 μg, about 50 μg or about 100 μg is used for each human injection. The timing of injection can vary significantly from once a day to once a year to once every 10 years. On any given day when an immunogen dose is given, this dose is higher than 1 μg per patient, usually higher than 10 μg per patient if adjuvant is also administered, and in the absence of adjuvant. Higher than 10 μg per patient, usually higher than 100 per patient. The mass of peptide present in this dosage can be used to calculate the amount of therapeutic agent used.

  One representative regimen consists of immunization following booster injections at 6 week intervals. The other regimen consists of immunization according to booster injections after 1, 2 and 12 months. Other regimens result in injections every two months for life. Alternatively, booster injections can be based on irregular criteria as indicated by immune response monitoring.

  For passive immunization with antibodies, this dosage ranges from about 0.0001 mg / kg host body weight to about 100 mg / kg body weight, more usually from about 0.01 mg / kg host body weight to about 100 mg / kg body weight. The range is 5 mg.

  The therapeutic agent for inducing an immune response can be any means suitable for prophylactic and / or therapeutic treatment (e.g. parenteral means, topical means, intravenous means, oral means, subcutaneous means, intraperitoneal means, Nasal or intramuscular means). The most typical route of administration is subcutaneous, but other routes may be equally effective. The next most common is intramuscular injection. This type of injection is most typically performed in the arm or leg muscles. Intravenous injections as well as intraperitoneal injections, intraarterial injections, intracranial injections or intradermal injections may also be effective in generating an immune response. In some methods, these therapeutic agents are injected directly into specific tissues where deposits are or can accumulate.

  The compositions of the invention can be administered in combination with other agents that are at least partially effective in the treatment of amyloidogenic diseases, if desired. In the case of Alzheimer's disease and Down syndrome, where amyloid deposition occurs in the brain, the therapeutic agents of the invention may also be administered with other agents that increase the passage of the compositions of the invention across the blood brain barrier.

  The immunogenic agents (eg, peptides) of the present invention are often administered in combination with an adjuvant. A variety of adjuvants can be used in combination with the immunogens of the present invention to elicit an immune response. Preferred adjuvants enhance the endogenous response to this immunogen without causing a conformational change in the immunogen that affects the qualitative form of the response. Preferred adjuvants include alum, 3 de-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211). QS21 is a triterpene glycoside or saponin isolated from the bark of a Quillaja Saponaria Molina tree found in South Africa (Kensil et al., Vaccine Design: The subpant Apovant P And Newman, Plenum Press, New York, 1995); and US Pat. No. 5,057,540). Other adjuvants are oil-in-water emulsions (eg squalene or peanut oil) optionally combined with an immunostimulant (eg monophosphoryl lipid A). See, for example, Stout et al. Engl. J. et al. Med. (1997) 336, 86-91. Another useful adjuvant is CpG described in Bioworld Today, November 15, 1998. Alternatively, the immunogen can be coupled to an adjuvant. For example, the lipopeptide version of this immunogen is described in Livingston, J. et al. Immunol. (1997) Vol. 159, pages 1383 to 1392, prepared by directly coupling palmitic acid or other lipids to the N-terminus of one or more peptides constituting the immunogen of the present invention. obtain. However, such coupling should not substantially change the conformation of the peptides that make up the immunogen so as to affect the nature of the immune response to the immunogen. Adjuvants can be administered with the active agent as a component of the therapeutic composition, or are administered separately from the administration of the therapeutic agent, prior to administration of the therapeutic agent, simultaneously with administration of the therapeutic agent, or after administration of the therapeutic agent. Can be done.

  A preferred class of adjuvants are aluminum salts (alum, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate). Such adjuvants can be used with or without other specific immunostimulants (eg, MPL or 3-DMP), QS21, polymeric amino acids or monomeric amino acids (eg, polyglutamic acid or polylysine) .

  Another class of adjuvants is oil-in-water emulsion formulations. Such adjuvants include other specific immunostimulants (eg, muramyl peptides (eg, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), -acetyl-normuramyl-L-alanyl). -D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2- (1'-2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy ) -Ethylamine (MTP-PE), N-acetylglucosaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (N-acetylglucaminemin-N-acetylmuramyl-L-Al-) D-isoglu-L-Ala-dipalmito y propylamide, DTP-DPP, theramide (TM), or other bacterial cell wall components, or without this immunostimulant, can be used for oil-in-water emulsions: (a) Model 110Y microfluidizer (microfluidics) 5% squalene, 0.5% Tween 80, and 0.5% Span 85 formulated into submicron particles using a microfluidizer such as Microfluidics, Newton, Mass. MF59 (Wo90 / 14837) (including various amounts of MTP-PE as required), (b) to produce microfluidized or larger particle size emulsions into submicron emulsions Vortex SAF containing 10% squalene, 0.4% Tween 80, 5% pluronic-block polymer L121 and thr-MDP, and (c) 2% squalene, 0.2% Tween 80, and monophosphoryl lipid A (MPL) Ribi ™ adjuvant system (RAS) comprising one or more bacterial cell wall components from the group consisting of trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferably MPL + CWS (Detox ™) ), (Ribi Immunochem, Hamilton, Mont.).

  Other classes of preferred adjuvants include saponin adjuvants (eg, Stimulons (QS21, Aquila, Worcester, Mass.)) Or particles generated therefrom (eg, ISCOM (immunostimulatory complex) and ISCOMATRIX) ). Other adjuvants include Freund's complete adjuvant (CFA) and incomplete Freund's adjuvant (IFA). Other adjuvants include cytokines (eg, interleukins (eg, IL-1, IL-2 and IL-12), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF)) and / or chemokines (eg, CXCL10 and CCL5) are included.

  The adjuvant can be administered with the immunogen as a single composition, or can be administered prior to, simultaneously with, or following administration of the immunogen. The immunogen and adjuvant can be packaged and supplied in the same vial or can be packaged in separate vials and mixed prior to use. The immunogen and adjuvant are generally packaged with a label indicating the intended therapeutic application. Where the immunogen and adjuvant are packaged separately, the package generally includes instructions for mixing prior to use. The choice of adjuvant and / or carrier depends on the stability of the vaccine containing the adjuvant, the route of administration, the administration schedule, the efficacy of the adjuvant for the species to be vaccinated, and in humans the pharmaceutically acceptable adjuvant is An adjuvant that is or may be approved for human administration by an appropriate regulatory agency. For example, Freund's complete adjuvant is not suitable for human administration. If desired, two or more different adjuvants can be used simultaneously. Preferred combinations include alum and MPL, alum and QS21, MPL and QS21, and alum, QS21 and MPL. Freund's incomplete adjuvant can also be used in combination with any of alum, QS21 and MPL and any combination thereof as needed (Chang et al., Advanced Drug Delivery Reviews Vol. 32, 173). 186 pages (1998)).

  The compositions of the present invention are often administered as pharmaceutical compositions consisting of various other pharmaceutically acceptable ingredients. See Remington's Pharmaceutical Sciences (15th Edition, Mack Publishing Company, Easton, Pa., 1980). The preferred form depends on the intended mode of administration and therapeutic application. These compositions are also defined as pharmaceutically acceptable excipients that are commonly used to formulate pharmaceutical compositions for administration to animals or humans, depending on the desired formulation. Non-toxic carriers or diluents. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate buffered saline, Ringer's solution, dextrose solution and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. However, some reagents suitable for animal administration such as Freund's complete adjuvant are generally not included in compositions for human use.

  Pharmaceutical compositions are also large and slowly metabolized macromolecules (eg, proteins, polysaccharides, polylactic acids, polyglycolic acids and copolymers (eg, latex functionalized sepharose, agarose, cellulose, etc.), polymeric amino acids , Amino acid copolymers, and lipid aggregates (eg, oil droplets or liposomes) In addition, these carriers can function as immunostimulants (ie, adjuvants).

  For parenteral administration, the compositions of the invention may be used as an injectable dosage of a solution or suspension of a substance in a physiologically acceptable diluent as a sterile liquid (eg, water, oil, saline, glycerose or Ethanol) and can be administered with a pharmaceutical carrier.

  Auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like may be present in the composition. Other ingredients of the pharmaceutical composition are those of petroleum, animal, vegetable or synthetic origin (eg peanut oil, soybean oil and mineral oil). In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

  The compositions can be prepared as injectables, either as liquid solutions or suspensions, and solid forms suitable for making solutions or suspensions in liquid solvents prior to injection can also be prepared. The preparation can also be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide or copolymers for enhanced adjuvant effects as discussed above. See Langer, Science (1990), 249, 1527 and Hanes, Advanced Drug Delivery Reviews (1997), 28, 97-119. The compositions of the present invention can be administered in the form of depot injections or implant preparations that can be formulated in a manner to allow sustained or pulsatile release of the active ingredient.

Additional formulations suitable for other modes of administration include oral, nasal and pulmonary formulations, suppositories, and transdermal administration.
For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides, and such suppositories are in the range of 0.5% to about 10%, such as in the range of about 1% to about 2%. It can be formed from a mixture containing the active ingredients within. Oral formulations include excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to 95% active ingredient, eg about 25% to about 70% active ingredient. Can be included.

  Topical application can result in transdermal or intradermal delivery. Topical administration can be facilitated by co-administration of the composition with cholera toxin or a detoxified derivative or subunits thereof, or other similar bacterial toxins. See Glenn et al., Nature (1998) 391, 851. Co-administration can be achieved by using these components as a mixture or as linked molecules obtained by chemical cross-linking or expression as a fusion protein.

  Alternatively, transdermal delivery can be achieved using skin passages or using transferosomes. For example, Paul et al., Eur. J. et al. Immunol. (1995) 25, 3521-24; Cevec et al., Biochem. Biophys. Acta (1998) 1368, 201-15.

  The present invention provides a method for detecting an immune response against an amyloid oligomeric intermediate in a patient suffering from or susceptible to an amyloid disease such as AD. These methods are particularly useful for monitoring the course of treatment administered to a patient. These methods can be used to monitor both therapeutic treatment for symptomatic patients and prophylactic treatment for asymptomatic patients.

  Some methods include determining a baseline value of the immune response in the patient and administering this value to a value for the post-treatment immune response before administering the formulation of the composition. To do. A significant increase in the value of the immune response (ie, an increase expressed as one standard deviation from the mean of such measurements and higher than the standard limit of experimental error in repeated measurements of the same sample) is positive Indicates a therapeutic outcome (ie, administration of the composition has achieved or enhanced an immune response). If the value for the immune response does not change significantly or decreases, a negative treatment outcome is indicated. In general, patients undergoing the first course of treatment with the composition are expected to show an increase in the immune response with continuous dosing eventually reaching stagnation. Administration of the composition is generally continued with an increased immune response.

The arrival of the stagnation period is an indication that this treatment can be interrupted or reduced in dosage or frequency.
In other methods, control values (ie, mean and standard deviation) of the immune response are determined for the control population. Typically, individuals in the control population are those who have not previously received treatment. The value of the immune response measured in the patient after administering the therapeutic composition is then compared to a control value. A significant increase compared to the control value (eg, greater than 1 standard deviation from the mean) indicates a positive treatment outcome. A lack or decrease in significant increase indicates a negative treatment outcome.

Administration of the composition is generally continued while the immune response increases relative to the control value.
As before, reaching the stagnation period relative to the control value is an indication that treatment administration can be interrupted or reduced in dosage or frequency.

  In other methods, control values (eg, mean and standard deviation) of the immune response are determined from a control population of individuals who have received treatment with the therapeutic composition and whose immune response has reached stagnation in response to treatment. Is done. Measurements of the immune response in the patient are compared to control values. If the measured level in the patient does not differ significantly from the control value (eg, not greater than one standard deviation), treatment can be interrupted. If the level in the patient is significantly lower than the control value, continued administration of the composition is warranted. If the level in the patient remains below the control value, a change in the treatment regimen (eg, use of a different adjuvant) may be indicated.

  In other methods, patients who are not currently receiving treatment but who have previously undergone a course of treatment are observed for an immune response to determine whether resumption of treatment is necessary. The measure of immune response in this patient can be compared to the value of the immune response previously achieved in this patient after a previous course of treatment. A significant decrease over previous measurements (eg, greater than the typical limit of error in repeated measurements of the same sample) is an indication that treatment can be resumed. Alternatively, the value measured in the patient can be compared to a control value (mean + standard deviation) determined in the patient population after undergoing the course of treatment.

  Alternatively, the value measured in the patient is a control value in a population of patients who are treated prophylactically and still have no symptoms of the disease, or a control in a population of patients who are therapeutically treated and show improved disease characteristics It can be compared with the value. In all of these cases, a significant decrease relative to the control value (eg, a decrease greater than one standard deviation) is an indication that treatment should be resumed in the patient.

  The tissue sample for analysis is generally blood, plasma, serum, mucus or cerebrospinal fluid from the patient. This sample can be analyzed for indicators of immune response to amyloid peptide aggregates or amyloid peptide aggregate mimics. This immune response can be determined, for example, from the presence of antibodies or T cells that specifically bind to amyloid peptide aggregates or amyloid peptide aggregate mimics. An ELISA method for detecting antibodies specific for the composition is described in the Examples section.

The present invention further provides a diagnostic kit for carrying out the above diagnostic method. In general, such kits include a composition that specifically binds to an antibody against the oligomeric intermediate or a composition that reacts with T cells specific for the oligomeric intermediate. The kit can also include a label. For detection of antibodies against amyloid peptide aggregates, this label is generally in the form of a labeled anti-idiotype antibody. For antibody detection, the composition can be supplied pre-bound to a solid phase, such as a microtiter dish well. In the case of detection of reactive T cells, this label can be supplied with ³ H-thymidine to measure the proliferative response. The kit also typically includes instructions for use of the kit. The instructions may include a chart or other corresponding regimen that correlates the level of label measured with the level of antibody to the oligomeric intermediate or the level of T cells reactive with the oligomeric intermediate.

Example Example 1
Molecular mimics of amyloid peptide aggregates were synthesized as follows.

Peptide synthesis:
Solid phase synthesis and purification of A40 (SEQ ID NO: 1), A42 (SEQ ID NO: 2), IAPP (SEQ ID NO: 3) and human prions 106-126 (SEQ ID NO: 4) was performed by D. Burdick et al. .) (1992) J Biol Chem Vol. 267, pages 546-54, performed by fluoren-9-ylmethoxycarbonyl chemistry using a continuous flow semi-automatic instrument. C-terminal thioesters by Fmoc chemistry were performed as substantially described in Inginito, R. et al. (1999) Journal of the American Chemical Society Vol. 121, pages 11369-11374. .

  For each peptide, 5 equivalents of the first amino acid ((Fmoc-Ala for A42) per gram of sulfurylbutyry-AM-PEGA resin (Novabiochem, San Diego, Calif.) In 10 mL of dichloromethane (DCM). -OH), (Fmoc-Val-OH for A40) and (Fmoc-Tyr (t-But) -OH for IAPP)) were added to manually couple the first amino acid to the sulfurylbuty-AM-PEGA resin. Followed by 10 equivalents of diisopropylethylamine (DIEA). The mixture was stirred at room temperature for 20 minutes and then cooled to -10 ° C to -20 ° C. 4.7 equivalents of ByBop (benzotriazol-1-yl-oxy-tris (pyrrolidino) phosphonium hexafluorophosphate) was added and the mixture was stirred at -10 ° C to -20 ° C for 8-9 hours. The substitution level is about 0.18-0.20 mmole / g, as determined using the Fmoc cleavage method by confirming coupling efficiency using the Kaiser test well known in the field of peptide synthesis. It was found. Acetylation was performed using acetic anhydride. The amino acid chain was extended by fluoren-9-ylmethoxycarbonyl chemistry using a continuous flow semi-automatic instrument. 100 mg of peptide was added in a synthesis vessel with N-methyl-2-pyrrolidone 5 × (NMP) 5.0 mL NMP, 185 μL i-Pr2EtN (1.1 mmol) and 400 μL iodoacetonitrile (previously in the dark with alumina basic filter Filtered on the bed). The reaction mixture was stirred on a rotary plate and in the dark for 24 hours. The resin was washed with 5 × NMP and 5 × DMF followed by 5 × CH 2 Cl 2 and then dried. 100 mg of resin was washed with 5 × THF, followed by the addition of 2.7 mL of THF. 2.7 mL of TMS-CH2N2 (50:50, v / v, hexane) was then added. After stirring for 2 hours, the resin was washed with 5 × 5 mL THF and 5 × 5 mL DMF.

  The resin was added to 120 μL of ethyl-3-mercaptopropionate and the mixture was stirred on a rotary plate for 24 hours. The resin was then filtered and washed with 3 × 3 ml DMF. The filtrate and washings were collected and rotary evaporated at 34 ° C. The yield was about 60%.

  The resulting peptide was deprotected using standard methods (TFA and scavenger) and purified by RP-HPLC. Its purity was confirmed by analytical RP-HPLC and electrospray mass spectrometry.

Human insulin, lysozyme, polyglutamine KKQ40KK and -synuclein were obtained from commercial sources or other sources.
A C-terminal thioester was added to each of these synthetic and commercially available peptides in a conventional manner.

Colloidal gold amyloid oligomer molecular mimic assembly:
Colloidal gold nanospheres (average diameter 5.3 nm) were purchased from Ted Pella, Inc., washed with 1M HCl and then washed 3 times with distilled water. These gold nanospheres were incubated for 3 hours in a solution of 0.2 mg / ml C-terminal thioester peptide (pH 5.0 to 5.5). The pH was then adjusted to 7.4 with 100 mM Tris (pH 8.0) (0.2% sodium azide).

  After incubating at room temperature for 6 hours, the antigen was recovered by centrifugation at 30,000 × G, 4 ° C., and 30 minutes, and washed with PBS (pH 7.6) three times to remove unincorporated peptide. And then dispersed in 0.02% sodium azide. The resulting micelle molecular mimics were analyzed by atomic force microscopy (AFM), circular dichroism spectroscopy, thioflavin T fluorescence, bis-ANS fluorescence and UV / visible spectroscopy, and the peptide monolayer on gold was , Confirmed to have the same secondary structure and conformation as the oligomeric amyloid intermediate representation in solution. This solution was stored at 4 ° C.

A collection of molecular mimetics is shown in FIG.
Example 2
Production of antibodies against the colloidal gold amyloid oligomer molecular mimic was performed as follows.

  In New Zealand white rabbits, Balb / C, C57 / Black6 mice and domestic dogs, approximately 08. A large amount of molecular mimetic produced as described in Example 1 was injected, corresponding to ~ 1.0 mg Aβ peptide. Gold-binding antigen was mixed with alum adjuvant, Freund's incomplete adjuvant, or no adjuvant (PBS only) prior to injection. Rabbits were immunized with 1 mL of antigen (0.08-0.1 mg peptide per rabbit dialyzed overnight at 4 ° C. against PBS). For the first injection, the same amount of antigen and Freund's complete adjuvant were used. For 11 subsequent injections, this antigen was mixed with Freund's incomplete adjuvant and each was injected at 2-week intervals. The animals were injected subcutaneously in small increments of 0.1 mL per checkerboard style site on the shoulder.

  Serum was collected by venipuncture. The IgG fraction is affinity purified on protein G-Sepharose beads, eluted in 0.2 M glycine (pH 2.2), neutralized to pH 7.4 with Tris buffer, then PBS (pH 7.4). Dialyzed against. An amyloid oligomer intermediate molecular mimetic is mixed with the IgG fraction, incubated for about 2 hours, and then washed, so that an intermediate aggregate-specific antibody (referred to as an oligomeric antibody) by adsorption on this molecular mimetic. Was purified. This oligomeric intermediate specific antibody was eluted in 0.2 M glycine (pH 2.2), then neutralized and dialyzed against PBS. The antibody was stored at 4 ° C or -70 ° C in PBS containing 0.02% sodium azide as a preservative.

  Polyclonal serum produced by vaccination of rabbits with this molecular mimic is specific for amyloid peptide aggregation intermediates and is not detectably reactive with soluble low molecular weight or fibrillar Aβ species (See Example 4). Surprisingly, anti-oligomer immunoreactivity to low molecular weight Aβ or Aβ fibrils was not observed for unfractionated sera even after boosting the rabbit 12 times, indicating that these molecular mimetics It shows that the immune response to the object is very specific.

Example 3
Describe the production of monomeric or low molecular weight aggregates, oligomeric intermediates and mature amyloid fibrils.

Preparation of Aβ monomers and low molecular weight aggregates:
Monomeric peptides and low molecular weight aggregates were prepared by dissolving 1.0 mg Aβ in 400 μL HFIP at room temperature. 100 μL of the resulting Aβ solution was added to 900 μL DD H ₂ O in a siliconized Eppendorf tube. After incubating at room temperature for 10-20 minutes, these samples were centrifuged at 14,000 × G for 15 minutes, and the supernatant fraction (pH 2.8-3.5) was transferred to a new siliconized tube. HFIP was evaporated in a gentle stream of ₂ for 5-10 minutes. These samples were then used immediately or fractionated by gel permeation to remove any fibrillar or oligomeric intermediates.

Preparation of Aβ oligomer intermediate:
Amyloid peptide aggregates were prepared by dissolving 1.0 mg Aβ in 400 μL HFIP for 10-20 minutes at room temperature. 100 μL of the resulting Aβ solution was added to 900 μL of DD H ₂ O in a siliconized Eppendorf tube. After incubating at room temperature for 10-20 minutes, these samples were centrifuged at 14,000 × G for 15 minutes and the supernatant fraction was transferred to a new siliconized tube and placed in a gentle stream of N ₂ for 5-10. HFIP was allowed to evaporate for a minute. These samples were agitated at 22 ° C. and 500 RPM for 24-48 hours using a Teflon coated micro stirrer bar. Ten μl aliquots were taken at 6-12 hour intervals for observation by AFM or EM. To prepare a high purity sample of the intermediate, residual trifluoroacetate ions were removed by lyophilization in 1 mM HCl followed by lyophilization in 50% acetonitrile.

  The agitation time required to obtain an optimal level of intermediate depends on factors that are obvious and subtle to those skilled in the art, including agitation speed and peptide concentration. The highest level of intermediate for Aβ was recovered after stirring between 6 hours and 3 days.

  The amount of oligomeric intermediates and monomers or low molecular weight aggregates were carefully observed using a Toso Haas TSK 300 gel permeation column or a Superose HR75 FPLC column. This intermediate was recovered at or near the void volume of the column from the monomer Aβ or low molecular weight Aβ eluting at or near the column volume.

  Purification of the intermediate from the fibrils is performed by centrifugation at 100,000 × G and 1 hour. Monomers or low molecular weight aggregates are removed by application of the supernatant to a gel permeation chromatography column. Intermediates are eluted near the void volume of the column, and monomers and low molecular weight aggregates are eluted near the containing volume and discarded.

Preparation of primordial fibrils:
Spherical oligomers were prepared as described above, then the same volume of PBS (pH 7.4) was added and stirred for 24 hours to produce spherical oligomer curved fibers.

Preparation of cyclic primordial fibrils:
For the preparation of cyclic primordial fibrils, oligomeric intermediates were prepared as described above. The sample is subjected to vigorous stirring while being dried by slow evaporation. To obtain substantially the same results, a few drops (less than 5% of the total volume) of hexane are added while stirring the sample. This is done 10 times with a 5 minute stirring period for each addition.

Preparation of fibrils:
The fibrils were separated into three different conditions (water each containing 0.02% sodium azide (pH 3.8-4.2), 10 mM Tris (pH 7.4), and 50 mM Tris 100 mM NaCl (pH 7.4)). Prepared below. The final peptide concentration of Aβ was 0.3-0.5 mg / ml (80-125 μM). These samples were stirred for 6-9 days at room temperature and 500 rpm with a Teflon-coated micro stirrer bar. Fibril formation was observed by thioflavin T fluorescence and UV light scattering. When fibril formation is complete, these solutions are centrifuged at 14,000 × G and 20 minutes, the fibril pellet is washed 3 × with double distilled water, and then resuspended in the desired buffer. did. The presence of mature fibril morphology and the absence of globular oligomeric intermediates and primordial fibrils were confirmed by AFM or negative staining EM.

Example 4
The specificity of the anti-oligomer-aggregating antibody was tested by screening SHSY5Y cell lysates for cellular proteins that react with the anti-oligomer antibody using dot blot analysis and ELISA assay.

Dot Blot Assay Monomers or low molecular weight aggregates, oligomeric intermediates and amyloid fibrils were prepared as described in Example 3 and each was immediately before use at a concentration of 0.5 mg / ml DD H ₂ O Dissolved in. 2 μl of each sample was poured onto a nitrocellulose membrane. The membrane was blocked for 1 hour at room temperature with 10% non-fat dry milk in Tris-buffered saline (TBS) (TBT-T) containing 0.01% Tween 20. The membrane was washed 3 times with TBS-T (5 minutes each) and then affinity purified anti-oligomer antibody (0.1 μg / ml in 3% BSA in TBS-T) or serum for 1 hour at room temperature. And incubated at (1: 1,000 dilution in 3% BSA TBS-T). Since higher concentrations of detergent have been shown to prevent detection of amyloid peptide aggregates by anti-oligomers, the concentration of Tween 20 is lower than one-tenth of the commonly used concentration. These membranes were washed 3 times with TBS-T (5 minutes each) and diluted 1: 10,000 in 3% BSA / TBS-T horseradish peroxidase-conjugated anti-rabbit IgG (Promega) And incubated at room temperature for 1 hour. These blots were washed 3 times with TBS-T and developed with an ECL chemiluminescence kit from Amersham-Pharmacia, Piscataway, NJ. For anti-oligomers, leave the same membrane at 65 ° C. and 45 minutes in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) as described above. Stripped, washed 5 times with TBS-T for 5 minutes, blocked with 10% nonfat dry milk, and immunodetected with 6E10. 6E10 is a known monoclonal antibody that detects amino acid residues 1 to 17 of human β-amyloid peptide.

As shown in FIG. 2, the following was applied to a nitrocellulose membrane and probed with anti-oligomer aggregates (anti-oligo):
1-soluble A40 oligomer aggregate intermediate;
2-soluble low molecular weight A40; and 3-A40 fibrils.

It can be seen in FIG. 2 that only the anti-oligomer recognizes soluble aggregated intermediates and 6E10 recognizes all A species.
ELISA Assay Samples were applied to 96 well plates and analyzed by ELISA using anti-oligomer aggregate antisera produced as described in Example 2. The analysis results for soluble low molecular weight A40 (A), soluble A40 oligomer (O), and A40 fibrils (■) are shown in FIG.

  Samples are diluted in coating buffer (0.1 M sodium bicarbonate, pH 9.6) and between 0 ng and 100 ng of each amyloid type in 100 μl buffer added to separate wells of a 96 well microplate did. The plates were incubated at 37 ° C. for 2 hours, washed 3 times with PBS containing 0.01% Tween 20 (PBS-T), and then blocked with 3% BSA TBS-T at 37 ° C. for 2 hours. The BSA used was IgG free (Sigma). The plates were then washed 3 times with PBS-T, 100 μl of anti-oligomer (diluted 1: 10,000 in 3% BSA / TBS-T) was added and incubated at 37 ° C. for 1 hour. The plates were washed 3 times with PBS-T and 100 μl horseradish peroxidase-conjugated anti-rabbit IgG (Promega, diluted 1: 10,000 in 3% BSA TBS-T) was added, Subsequently, it was allowed to stand at 37 ° C. for 1 hour. These plates were washed 3 times with PBS-T and using 3,3 ′, 5,5′-tetramethylbenzidine (TMB, KPL, KPL, Gaithersburg, Md.). Color was developed. The reaction was stopped with 100 μL of 1M HCl and the plates were read at 450 nm.

  From FIG. 3, it can be seen that only the amyloid peptide-aggregated A40 intermediate was recognized by the anti-oligomer serum, and the soluble low molecular weight A40 and A40 fibrils showed only background values.

Example 5
The kinetics of anti-oligomer immunoreactivity during fibril formation was analyzed by time point dot blot assay (FIG. 4).

  Spherical aggregates are not initially present in the newly solubilized solution of the denatured Aβ peptide and occur over time. Furthermore, the formation of spherical oligomers is known to precede the formation of curvilinear fibers or primordial fibrils. See, for example, Harper et al (1999) Biochemistry 38, 8972.

  FIG. 4 shows a time point dot blot in which A40 and A42 solutions were dissolved in HFIP, diluted to 56 μM Aβ, and incubated at 25 ° C. with stirring in 100 mM NaCl, 50 mM Tris (pH 7.4). The assay is shown. At the indicated times, aliquots were applied to nitrocellulose membranes and probed with anti-oligomer antibodies (upper panel), then stripped and reprobed with 6E10 (lower panel).

  For A42, immunoreactivity is observed at 6 hours and is maximal between 24 and 168 hours. Immunoreactivity is lost at 332 hours. The kinetics for A40 are similar to those of A42 except that the formation of the intermediate is delayed for about 18-24 hours (this is consistent with previous observations that A42 forms oligomers faster than A40). It is almost the same as theory. These samples were examined by electron microscopy to determine the morphology during this time course experiment. In the early stages of immunoreactivity, these samples mainly contained globular oligomers, but in later stages, it was confirmed that they mainly contained elongated “primitive fibrils”. This observation indicates that protofibrils and less developed intermediate amyloid forms present the same conformational epitope recognized by anti-oligomers.

Example 6
The dependence of peptide aggregate size on the appearance of anti-oligomer aggregate epitopes was determined by size exclusion chromatography as described in Soleham et al (1994) J Biol Chem Vol. 269, 28551. Were tested by fractionating.

The A40 oligomer solution was incubated at different conditions favoring a population of oligomers of various sizes. (−) A40 left for 3 days in DD H ₂ O (pH 2.5-4), (40) A40 left for 2 days in 50 mM Tris (pH 7.4) 100 mM NaCl, (− ---) A40 newly dissolved in 50 mM Tris (pH 7.4), (...) A40 left incubating in 50 mM Tris (pH 7.4) for 2 days. Peaks were collected and aliquots from each were doted onto nitrocellulose membranes and probed with anti-oligomer antibody and 6E10 antibody. The results are shown in FIG.

  A 40 kDa peptide aggregate that elutes at a position of about 40 kDa (which corresponds to the approximate size of the octamer) is recognized by the anti-oligomer. Peaks eluting at positions corresponding to tetramers, dimers and monomers do not show reactivity with anti-oligomers.

Example 7
The specificity of the anti-oligomer sera produced in Example 2 was analyzed by reactivity with other amyloidogenic proteins and peptides by ELISA. This includes analysis of fibrillar aggregates, low molecular weight oligomers and amyloid fibrils derived from -synuclein, islet amyloid IAPP, polyglutamine, lysozyme, human insulin and human prion peptides 106-126.

  Samples were applied to 96-well plates and analyzed by ELISA using an anti-oligomer antiserum (which was performed substantially as described in Example 4) (FIG. 6). Soluble low molecular weight oligomers (▲), amyloid peptide aggregates (◯) and A40 peptide (■) were analyzed for each amyloid type. Only amyloid peptide aggregates are recognized by anti-oligomers, soluble low molecular weight oligomers and fibrils give only background values. The amyloid type is shown at the top of each panel in FIG.

  These results indicate that anti-oligomers recognize the unique common conformational structural features of the polypeptide backbone in amyloid peptide aggregates and are not defined by unique amino acid primary sequences.

Example 8
The ability of anti-oligomer antibodies to inhibit neurotoxicity in cell cultures was tested.
Inhibition of cytotoxicity of amyloid peptide aggregates by anti-oligomer antibodies is measured using MTT reduction and LDH release toxicity assays in human neuroblastoma SH-SY5Y cells.

For the MTT reduction assay, SH-SY5Y human neuroblastoma cells were 5% in DMEM containing 10 mM HEPES, 10% fetal calf serum, 4 mM glutamine, penicillin (200 units / ml) and streptomycin (200 μg / ml). Stored in CO ₂ and at 37 ° C. The medium was changed every 2 days. Prior to use, cells were differentiated in serum-free DMEM medium containing N2 feed and 1 × 10 ⁻⁵ M all-trans retinoic acid. Cells were plated at 10,000 cells / well in 96 well plates and grown overnight. The medium was removed and the amyloid form to be tested was added to 80 μl of fresh medium without phenol red. After incubation at 37 ° C. for 4 hours, the cells were analyzed using an MTT toxicology kit (Tox-1, Sigma) according to the manufacturer's instructions.

  For the LDH release assay, SH-SY5Y cells were prepared and treated in amyloid form as described above. After 8 hours at 37 ° C., LDH assays were performed using an LDH toxicology kit (Tox-7, Sigma) according to the manufacturer's instructions.

  FIG. 7 shows the toxicity of A40 and A42 oligomeric intermediates and the inhibition of toxicity by anti-oligomers utilizing MTT reduction for A40 and A42 fibrils. Samples were added for 30 minutes in the presence of an excess of affinity-purified anti-oligomer antibody (white bar), in the absence (black bar), or in the presence of the same amount of non-immune rabbit IgG (hatched bar). Incubated in advance and then analyzed for cytotoxicity at a final concentration of 2.5 mM. Anti-oligomers are effective in substantially reducing the toxicity of oligomeric intermediates (oligos). The fibril form is initially substantially non-toxic and is not substantially affected by the anti-oligomer.

  -By anti-oligomers utilizing MTT reduction of toxicity of other amyloid peptide aggregates, including amyloid peptide aggregates derived from synuclein, islet amyloid polypeptide (IAPP), polyglutamine, lysozyme, human insulin and human prion peptides 106-126 Inhibition is shown in FIG. FIG. 8 also shows the cytotoxicity measurements for soluble low molecular weight oligomers and fibrils (All Sol. And All Fib.) And their reduction by anti-oligomers in the total average measurement for all amyloid types tested. .

  LDH release assay in human neuroblastoma SH-SY5Y cells for amyloid peptide aggregates of A40, A42, synuclein, islet amyloid polypeptide (IAPP), polyglutamine, lysozyme, human insulin and human prion peptide 106-126 Inhibition of toxicity by anti-oligomer antibodies as measured in use is shown in FIGS. 9a and 9b. Soluble oligomer samples were prepared in the presence of an excess of affinity purified anti-oligomer antibody (white bar), in the absence (black bar), or in the presence of the same amount of nonimmune rabbit IgG (hatched bar). Pre-incubated for minutes and then analyzed for cytotoxicity at a final concentration of 2.5 mM. FIGS. 9a and 9b also show cytotoxicity measurements for soluble low molecular weight oligomers and fibrils (All Sol. And All Fib.) And their reduction by anti-oligomers in the total average measurement for all amyloid types tested. Indicates. FIG. 9b has a clear purpose and shows the white and black bars shown in FIG. 9b.

From this data it is clear that each amyloid peptide aggregate type tested results in inhibition of toxicity by anti-oligomer antibodies.
All tested amyloid amyloid peptide aggregates show significant toxicity, and this toxicity is eliminated by anti-oligomers, indicating that amyloid peptide aggregates have a common structure that can mediate toxicity by a common mechanism. Indicates sharing. Anti-oligomers are also shown to be effective antibodies for binding to and reducing the toxicity of a general class of amyloid oligomer intermediates.

Example 9
The specificity of the anti-oligomer was tested by dot blot analysis and ELISA assay.

Dot blot analysis:
Soluble SH-SY5Y cell lysate (2.8 μg) was mixed with 0 ng, 6 ng, 12 ng, 25 ng, 50 ng or 100 ng of A42 amyloid peptide aggregates. These samples were tested by a dot blot assay performed substantially as described in Example 4 (Figure 10).

  In the absence of added amyloid peptide aggregates, no anti-oligomer immunoreactivity is observed in cell lysates. Upper row: amyloid peptide aggregates incubated with cell lysate in the absence of protease inhibitor cocktail. Bottom row: amyloid peptide aggregates incubated with cell lysate in the presence of protease inhibitor cocktail. There is a significant increase in detectable amyloid peptide aggregates in the presence of protease inhibitors.

ELISA assay:
Soluble SH-SY5Y cell lysate (20 μg) was mixed with increasing amounts of A42, A40 and -synuclein amyloid peptide aggregates and subjected to an ELISA assay performed substantially as described in Example 4. (FIG. 11). As little as 0.75 ng amyloid peptide aggregates are detected above the background of cell lysate in the absence of amyloid peptide aggregates.

  As in this dot blot analysis, detection of added soluble oligomers when mixed with cell lysate is dependent on the presence of protease inhibitors. This indicates that soluble amyloid peptide aggregates are sensitive to proteolysis as previously reported (Walsh et al. (2002) Nature 416, 535-539). .

  The unfractionated serum produced in response to repeated immunization with this molecular mimic is remarkably specific for the pathological micelle conformation of the amyloidogenic peptide. This suggests that it may provide a means for vaccine development that avoids the undesirable inflammatory side effects observed for vaccination with A. This is because this vaccine specifically targets intermediates without reactivity to monomeric Aβ or fibril deposition (Hardy, Dee, J. Selkoe (2002). Science 297, 353-356). The result that all soluble amyloid oligomers tested are recognized by this antibody suggests that vaccines against this epitope may be an effective therapeutic approach for a wide range of amyloid diseases.

Example 10
Diagnostic method using ELISA assay:
Cerebrospinal fluid samples are serially diluted at a 2-fold dilution in coating buffer (0.1 M sodium bicarbonate, pH 9.6). 100 μl of sample was added to a well of a 96-well microplate, incubated at 37 ° C. for 2 hours, washed 3 times with PBS containing 0.01% Tween 20 (PBS-T), and then 3% BSA TBS- Block with T for 2 hours at 37 ° C. The BSA used is IgG free (Sigma). The plates are then washed 3 times with PBS-T, 100 μl of anti-oligomer (diluted 1: 10,000 in 3% BSA / TBS-T) is added and incubated at 37 ° C. for 1 hour. The plates were washed 3 times with PBS-T and 100 μl of horseradish peroxidase-conjugated anti-rabbit IgG (1: 10,000 dilution in Promega 3% BSA TBS-T) was added, 37 Incubate at ℃ for 1 hour. These plates were washed 3 times with PBS-T and using 3,3 ′, 5,5′-tetramethylbenzidine (TMB, KPL, KPL, Gaithersburg, Md.). Let the color develop. The reaction is stopped with 100 μL of 1M HCl and the plates are read at 450 nm. Anti-oligomer binding to the wells of the ELISA plate indicates the presence of an amyloid oligomer intermediate.

Example 11
Methods for assessing the efficacy of treatment methods:
Oligomer specific antibodies can be utilized in screening for drugs and therapeutic agents that inhibit the formation of amyloid oligomeric intermediates or cause degradation or dissociation of such oligomeric intermediates. To screen for drugs that inhibit amyloid oligomer intermediate formation, the test compound or drug is incubated with the amyloid peptide under conditions in which the amyloid oligomer intermediate forms in the absence of any inhibitory effect. This mixture is analyzed by an ELISA plate that determines the amount of amyloid oligomer intermediate formed, substantially as described in Example 4. In order to test for compounds that degrade or dissociate the oligomeric intermediate, the preformed oligomeric intermediate is mixed with the test drug or compound and the mixture is analyzed by ELISA to determine the amount of oligomeric intermediate present. Inhibitory compounds produce lower amounts of amyloid oligomers, which are detected by anti-oligomer antibodies in this assay.

  Although the foregoing invention has been described in detail for purposes of clarity of understanding, it will be apparent that certain modifications may be practiced within the scope of the appended claims. All publications and patent documents cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each were individually indicated.

The figure which shows the aggregate | assembly of the synthetic antigen of this invention. The figure which shows the result of the dot-blot assay which spotted Aβ low molecular weight oligomer, Aβ high molecular weight oligomer, and Aβ fibril on a nitrocellulose membrane, and probed with anti-oligomer antibody and 6E10. The figure which shows the result of the ELISA assay which analyzed A (beta) low molecular weight oligomer, A (beta) high molecular weight oligomer, and A (beta) fibril for anti-oligomer specificity. FIG. 4 shows the results of a dot blot assay demonstrating the formation of Aβ high molecular weight oligomers from Aβ low molecular weight oligomers and subsequent Aβ fibril formation from Aβ high molecular weight oligomers over time. The figure which shows the molecular sizing of the A (beta) assembly produced in the different solution as determined with time by dot blot analysis of the fraction eluted from the gel filtration column. The figure which shows the result of the ELISA assay which analyzed the various low molecular weight amyloid aggregate, the high molecular weight amyloid aggregate, and the amyloid fibril for anti-oligomer specificity. FIG. 5 shows the reduction in cytotoxicity of A40 and A42 fibrils (Fib.) And A40 and A42 high molecular weight aggregates (oligos) by anti-oligomer antibodies using the MTT reduction assay. A40, A2,-synuclein, islet amyloid polypeptide (IAPP), polyglutamine, lysozyme, human insulin and human prion peptides 106-126 low molecular weight aggregates (Sol.), High with anti-oligomeric antibodies using MTT reduction assay FIG. 5 shows a reduction in the cytotoxicity of molecular weight aggregates and fibrils (Fib.). A40, A42, synuclein, islet amyloid polypeptide (IAPP), polyglutamine, lysozyme, human insulin and human prion peptides 106-126 low molecular weight aggregates (Sol.), High by anti-oligomeric antibodies using LDH release assay FIG. 5 shows a reduction in the cytotoxicity of molecular weight aggregates and fibrils (Fib.). A40, A42, synuclein, islet amyloid polypeptide (IAPP), polyglutamine, lysozyme, human insulin and human prion peptides 106-126 low molecular weight aggregates (Sol.), High by anti-oligomeric antibodies using LDH release assay FIG. 5 shows a reduction in the cytotoxicity of molecular weight aggregates and fibrils (Fib.). FIG. 5 shows a dot blot analysis demonstrating the specificity of anti-oligomer antibodies in cell extracts. FIG. 5 shows an ELISA assay that demonstrates the specificity of anti-oligomer antibodies in cell extracts.

Claims (180)

An isolated composition comprising a conformational epitope of a primordial fibril aggregate, wherein the primordial fibril aggregate is formed in a) a human or an animal, and b) contributes to amyloid fibril formation . The composition according to claim 1, wherein the composition is a synthetic product. The composition of claim 1, wherein the composition comprises a peptide. 4. The composition of claim 3, wherein the peptide is conformationally constrained. The peptide is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and mixtures thereof. The composition according to claim 3. 4. The composition of claim 3, wherein the peptide is SEQ ID NO: 1. The composition of claim 1, wherein the composition is supported by a surface. The composition of claim 7, wherein the surface is curved or flat. The composition of claim 7, wherein the surface comprises a solid. 8. The composition of claim 7, wherein the surface comprises a film, particle or sheet surface. The composition of claim 7, wherein the surface comprises a protein. The composition of claim 11, wherein the protein comprises an α-pleated sheet. The composition according to claim 7, wherein the composition is bound to the support surface. 8. The composition of claim 7, wherein the composition is chemically bonded to the support surface. The composition according to claim 14, wherein the chemical bond is a covalent bond. 8. The support of claim 7, wherein the support comprises a material selected from the group consisting of gold, zinc, cadmium, tin, titanium, silver, selenium, gallium, indium, arsenic, silicon, mixtures thereof, and combinations thereof. Composition. The composition of claim 1, wherein the protofibrillar aggregate has a molecular weight in the range of about 1 kDa to about 100,000,000 kDa. 2. The composition of claim 1, wherein the primordial fibril aggregate comprises 5 or more monomers. The composition of claim 1 wherein the primordial fibril aggregate comprises 8 monomers. The composition of claim 1, wherein the amyloid peptide monomer is substantially free of the epitope. 2. The composition of claim 1, wherein amyloid fibrils are substantially free of the epitope. 2. The composition of claim 1, wherein the primordial fibril aggregate comprises a toxic species. 2. The composition of claim 1, wherein the primordial fibril aggregate is present in a human or animal having a disease characterized by amyloid deposition. The disease is Alzheimer's disease, early-onset Alzheimer's disease associated with Down syndrome, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, and spongiform encephalopathy including mad cow disease, sheep scrapie and mink spongiform encephalopathy , Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, Gerstmann-Streisler-Scheinker syndrome, Kuru disease, fatal familial insomnia, chronic wasting syndrome, familial amyloid polyneuropathy 24. A composition according to claim 23 selected from the group consisting of cerebral disease, frontotemporal dementia, type II diabetes, systemic amyloidosis, serum amyloidosis, British familial dementia, Danish familial dementia, macular degeneration and cerebrovascular amyloidosis . 24. The composition of claim 23, wherein the disease is Alzheimer's disease. The composition according to claim 1, wherein the composition is a pharmaceutical composition. The composition of claim 1, wherein the composition is a vaccine. An isolated composition comprising an epitope of a protofibrillar aggregate, wherein the fibrillar aggregate forms in a human or animal and contributes to amyloid fibril formation, said amyloid fibril comprising said epitope A substantially free composition. 29. The composition of claim 28, wherein the composition is a synthetic product. 30. The composition of claim 28, wherein the amyloid peptide monomer is substantially free of the epitope. 30. The composition of claim 28, wherein the composition comprises a peptide. 32. The composition of claim 31, wherein the peptide is conformationally constrained. The peptide is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and mixtures thereof. 32. The composition of claim 31. 32. The composition of claim 31, wherein the peptide is SEQ ID NO: 1. 30. The composition of claim 28, wherein the composition is supported by a surface. 36. The composition of claim 35, wherein the surface is curved or flat. 36. The composition of claim 35, wherein the surface comprises a solid. 36. The composition of claim 35, wherein the surface is a film, particle or sheet. 36. The composition of claim 35, wherein the surface comprises a protein. 40. The composition of claim 39, wherein the protein comprises an [alpha] -pleated sheet. 36. The composition of claim 35, wherein the composition is bound to the support surface. 36. The composition of claim 35, wherein the composition is chemically bonded to the support surface. 43. The composition of claim 42, wherein the chemical bond is a covalent bond. 36. The support of claim 35, wherein the support comprises a material selected from the group consisting of gold, zinc, cadmium, tin, titanium, silver, selenium, gallium, indium, arsenic, silicon, mixtures thereof, and combinations thereof. Composition. 30. The composition of claim 28, wherein the primordial fibril aggregate has a molecular weight in the range of about 1 kDa to about 100,000,000 kDa. 29. The composition of claim 28, wherein the primordial fibril aggregate comprises 5 monomers. 29. The composition of claim 28, wherein the protofibrillar aggregate comprises 8 monomers. 29. The composition of claim 28, wherein the protofibrillar aggregate comprises a toxic species. 29. The composition of claim 28, wherein the primordial fibril aggregate is present in a human or animal having a disease characterized by amyloid deposition. The disease is Alzheimer's disease, early onset Alzheimer's disease associated with Down's syndrome, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, and spongiform encephalopathy including mad cow disease, sheep scrapie and mink spongiform encephalopathy , Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, Gerstmann-Streisler-Scheinker syndrome, Kuru disease, fatal familial insomnia, chronic wasting syndrome, familial amyloid polyneuropathy 50. The composition of claim 49, selected from the group consisting of dystrophy, frontotemporal dementia, type II diabetes, systemic amyloidosis, serum amyloidosis, British familial dementia, Danish familial dementia, macular degeneration, and cerebrovascular amyloidosis . 50. The composition of claim 49, wherein the disease is Alzheimer's disease. 29. A synthetic composition according to claim 28, wherein the composition is a pharmaceutical composition. 30. The composition of claim 28, wherein the composition is a vaccine. A composition comprising an isolated antibody, wherein the antibody binds to a conformational epitope of a fibrillar aggregate that forms in a human or animal and contributes to amyloid fibril formation. 55. The composition of claim 54, wherein the antibody is effective to reduce toxicity of the protofibrillar aggregate. 55. The composition of claim 54, wherein the protofibrillar aggregate has a molecular weight in the range of about 1 kDa to about 100,000,000 kDa. 55. The composition of claim 54, wherein the protofibrillar aggregate comprises 5 monomers. 55. The composition of claim 54, wherein the primordial fibril aggregate comprises 8 monomers. 55. The composition of claim 54, wherein the amyloid peptide monomer is substantially free of the conformational epitope. 55. The composition of claim 54, wherein amyloid fibrils are substantially free of the epitope. 55. The composition of claim 54, wherein the protofibrillar aggregate comprises a toxic species. 55. The composition of claim 54, wherein the primordial fibril aggregate is present in a human or animal having a disease characterized by amyloid deposition. The disease is Alzheimer's disease, early-onset Alzheimer's disease associated with Down syndrome, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, and spongiform encephalopathy including mad cow disease, sheep scrapie and mink spongiform encephalopathy , Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, Gerstmann-Streisler-Scheinker syndrome, Kuru disease, fatal familial insomnia, chronic wasting syndrome, familial amyloid polyneuropathy 63. A composition according to claim 62 selected from the group consisting of dystrophy, frontotemporal dementia, type II diabetes, systemic amyloidosis, serum amyloidosis, British familial dementia, Danish familial dementia, macular degeneration and cerebrovascular amyloidosis . 64. The composition of claim 62, wherein the disease is Alzheimer's disease. 55. The composition of claim 54, wherein the composition is a pharmaceutical composition. A composition comprising an isolated antibody, wherein the antibody binds to an epitope of a fibrillar aggregate that forms in a human or animal and contributes to amyloid fibril formation, the amyloid fibrils binding the epitope. A substantially free composition. 68. The composition of claim 66, wherein the protofibrillar aggregate comprises a toxic species. 68. The composition of claim 66, wherein amyloid peptide monomer is substantially free of the epitope. 68. The composition of claim 66, wherein the antibody is effective to reduce toxicity of the protofibrillar aggregate. 68. The composition of claim 66, wherein the protofibrillar aggregate has a molecular weight in the range of about 1 kDa to about 100,000,000 kDa. 68. The composition of claim 66, wherein the primordial fibril aggregate comprises 5 monomers. 68. The composition of claim 66, wherein the primordial fibril aggregate comprises 8 monomers. 68. The composition of claim 66, wherein the primordial fibril aggregate is present in a human or animal having a disease characterized by amyloid deposition. The disease is Alzheimer's disease, early onset Alzheimer's disease associated with Down's syndrome, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, and spongiform encephalopathy including mad cow disease, sheep scrapie and mink spongiform encephalopathy , Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, Gerstmann-Streisler-Scheinker syndrome, Kuru disease, fatal familial insomnia, chronic wasting syndrome, familial amyloid polyneuropathy 74. A composition according to claim 73 selected from the group consisting of cerebral disease, frontotemporal dementia, type II diabetes, systemic amyloidosis, serum amyloidosis, British familial dementia, Danish familial dementia, macular degeneration and cerebrovascular amyloidosis . 74. The composition of claim 73, wherein the disease is Alzheimer's disease. 68. The composition of claim 66, wherein the composition is a pharmaceutical composition. A method of preventing or treating a disease or condition in a human or animal subject, wherein the disease or condition is characterized by the presence of amyloid deposits, the method comprising:
A. Administering to the subject a therapeutically effective or prophylactic amount of a composition comprising a conformational epitope of a fibrillar aggregate that forms in a human or animal and contributes to amyloid fibril formation. 78. The method of claim 77, wherein step A comprises inducing an immune response against the conformational epitope. 78. The method of claim 77, wherein the composition comprises a peptide component. 80. The method of claim 79, wherein the peptide is conformationally constrained. The peptide is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and mixtures thereof. 80. The method of claim 79. 80. The method of claim 79, wherein the peptide is SEQ ID NO: 1. 78. The method of claim 77, wherein the composition is supported by a surface. 84. The method of claim 83, wherein the surface is curved or flat. 84. The method of claim 83, wherein the surface comprises a solid. 84. The method of claim 83, wherein the surface is a film, particle or sheet. 84. The method of claim 83, wherein the surface comprises a protein. 90. The method of claim 86, wherein the protein comprises an [alpha] -pleated sheet. 84. The method of claim 83, wherein the composition is bound to the support surface. 84. The method of claim 83, wherein the composition is chemically bonded to the support surface. The method of claim 90, wherein the chemical bond is a covalent bond. 84. The support of claim 83, wherein the support comprises a material selected from the group consisting of gold, zinc, cadmium, tin, titanium, silver, selenium, gallium, indium, arsenic, silicon, mixtures thereof, and combinations thereof. Method. 78. The method of claim 77, wherein the protofibrillar aggregate has a molecular weight in the range of about 1 kDa to about 100,000,000 kDa. 78. The method of claim 77, wherein the protofibrillar aggregate comprises 5 monomers. 78. The method of claim 77, wherein the primordial fibril aggregate comprises 8 monomers. 78. The method of claim 77, wherein amyloid peptide monomer is substantially free of said epitope. 78. The method of claim 77, wherein amyloid fibrils are substantially free of the epitope. 78. The method of claim 77, wherein the primordial fibril aggregate comprises a toxic species. 78. The method of claim 77, wherein the protofibrillar aggregate is present in a human or animal having a disease characterized by amyloid deposition. The disease is Alzheimer's disease, early onset Alzheimer's disease associated with Down's syndrome, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, and spongiform encephalopathy including mad cow disease, sheep scrapie and mink spongiform encephalopathy , Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, Gerstmann-Streisler-Scheinker syndrome, Kuru disease, fatal familial insomnia, chronic wasting syndrome, familial amyloid polyneuropathy 100. The method of claim 99, selected from the group consisting of dystrophy, frontotemporal dementia, type II diabetes, systemic amyloidosis, serum amyloidosis, British familial dementia, Danish familial dementia, macular degeneration, and cerebrovascular amyloidosis. 100. The composition of claim 99, wherein the disease is Alzheimer's disease. 78. The composition of claim 77, wherein the composition is a vaccine. A method for preventing or treating a disease or condition characterized by amyloid deposition in a human or animal comprising:
A. Administering to the subject a therapeutically effective or prophylactic amount of a composition comprising an epitope of a fibrillar aggregate that forms in humans or animals and contributes to amyloid fibril formation;
The method wherein the amyloid fibril is substantially free of the epitope. 104. The method of claim 103, wherein step A comprises inducing an immune response against the conformational epitope. 104. The method of claim 103, wherein the amyloid peptide monomer is substantially free of the epitope. 104. The method of claim 103, wherein the composition comprises a peptide component. 107. The method of claim 106, wherein the peptide is conformationally constrained. The peptide is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and mixtures thereof. 107. The method of claim 106. 107. The method of claim 106, wherein the peptide is SEQ ID NO: 1. 104. The method of claim 103, wherein the composition is supported by a surface. 111. The method of claim 110, wherein the surface is curved or flat. 111. The method of claim 110, wherein the surface comprises a solid. 111. The method of claim 110, wherein the surface is a film, particle or sheet. 111. The method of claim 110, wherein the surface comprises a protein. 115. The method of claim 114, wherein the protein comprises an [alpha] -pleated sheet. 111. The method of claim 110, wherein the composition is bound to the support. 111. The method of claim 110, wherein the composition is chemically bonded to the support. 118. The method of claim 117, wherein the chemical bond is a covalent bond. 111. The support of claim 110, wherein the support comprises a material selected from the group consisting of gold, zinc, cadmium, tin, titanium, silver, selenium, gallium, indium, arsenic, silicon, mixtures thereof, and combinations thereof. Method. 104. The method of claim 103, wherein amyloid fibrils are substantially free of the epitope. 104. The method of claim 103, wherein the fibrillar aggregate has a molecular weight in the range of about 1 kDa to about 100,000,000 kDa. 104. The method of claim 103, wherein the primordial fibril aggregate comprises 5 monomers. 104. The method of claim 103, wherein the primordial fibril aggregate comprises 8 monomers. 104. The method of claim 103, wherein the protofibrillar aggregate comprises a toxic species. 104. The method of claim 103, wherein the primordial fibril aggregate is present in a human or animal having a disease characterized by amyloid deposition. The disease is Alzheimer's disease, early-onset Alzheimer's disease associated with Down syndrome, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, and spongiform encephalopathy including mad cow disease, sheep scrapie and mink spongiform encephalopathy , Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, Gerstman-Streisler-Scheinker syndrome, Kuru disease, fatal familial insomnia, chronic wasting syndrome, familial amyloid polyneuropathy 129. The method of claim 125, selected from the group consisting of symptom, frontotemporal dementia, type II diabetes, systemic amyloidosis, serum amyloidosis, British familial dementia, Danish familial dementia, macular degeneration, and cerebrovascular amyloidosis. 126. The method of claim 125, wherein the disease is Alzheimer's disease. 104. The method of claim 103, wherein the composition is a vaccine. A method of preventing or treating a disease or condition characterized by amyloid deposition in a human or animal subject comprising:
A. A method comprising binding an antibody to a conformational epitope of a fibrillar aggregate that forms in a human or animal and contributes to fibril formation. 129. The method of claim 129, wherein step A comprises administering to the subject a therapeutically effective or prophylactic amount of the antibody. 129. The method of claim 129, wherein said protofibrillar aggregate comprises a toxic species. 132. The method of claim 131, wherein said antibody is effective in reducing toxicity of said protofibrillar aggregate. 129. The method of claim 129, wherein the protofibrillar aggregate has a molecular weight in the range of about 1 kDa to about 100,000,000 kDa. 129. The method of claim 129, wherein the protofibrillar aggregate comprises 5 monomers. 129. The method of claim 129, wherein the protofibrillar aggregate comprises 8 monomers. 129. The method of claim 129, wherein amyloid peptide monomer is substantially free of said epitope. 129. The method of claim 129, wherein amyloid fibrils are substantially free of said epitope. 129. The method of claim 129, wherein said primordial fibril aggregate is present in a human or animal having a disease characterized by amyloid deposition. The disease is Alzheimer's disease, early-onset Alzheimer's disease associated with Down syndrome, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, and spongiform encephalopathy including mad cow disease, sheep scrapie and mink spongiform encephalopathy , Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, Gerstman-Streisler-Scheinker syndrome, Kuru disease, fatal familial insomnia, chronic wasting syndrome, familial amyloid polyneuropathy 138. The method of claim 138, selected from the group consisting of dystrophy, frontotemporal dementia, type II diabetes, systemic amyloidosis, serum amyloidosis, British familial dementia, Danish familial dementia, macular degeneration, and cerebrovascular amyloidosis. 138. The method of claim 138, wherein the disease is Alzheimer's disease. The composition is intrathecal, intrathecal, oral, transdermal, pulmonary, intravenous, subcutaneous, nasal, intraarterial, intracranial, intradermal, intraperitoneal 132. The method of claim 129, wherein the method is administered by a method selected from the group consisting of: intramuscular administration, rectal administration, and buccal administration. A method of preventing or treating a disease or condition characterized by amyloid deposition in a human or animal subject comprising:
A. Binding the isolated antibody to an epitope of a fibrillar aggregate that forms in a human or animal and contributes to amyloid fibril formation;
The method wherein the amyloid fibril is substantially free of the epitope. 143. The method of claim 142, wherein step A comprises administering to the subject a therapeutically effective or prophylactic amount of antibody. 143. The method of claim 142, wherein the protofibrillar aggregate has a molecular weight in the range of about 1 kDa to about 100,000,000 kDa. 143. The method of claim 142, wherein the primordial fibril aggregate comprises 5 monomers. 143. The method of claim 142, wherein the fibrillar aggregate comprises 8 monomers. 143. The method of claim 142, wherein the protofibrillar aggregate comprises a toxic species. 143. The method of claim 142, wherein said antibody is effective in reducing toxicity of said protofibrillar aggregate. 143. The method of claim 142, wherein amyloid fibrils are substantially free of the epitope. 143. The method of claim 142, wherein the protofibrillar aggregate comprises a toxic species. 143. The method of claim 142, wherein the protofibrillar aggregate is present in a human or animal having a disease characterized by amyloid deposition. The disease is Alzheimer's disease, early-onset Alzheimer's disease associated with Down syndrome, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, and spongiform encephalopathy including mad cow disease, sheep scrapie and mink spongiform encephalopathy , Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, Gerstman-Streisler-Scheinker syndrome, Kuru disease, fatal familial insomnia, chronic wasting syndrome, familial amyloid polyneuropathy 153. The method of claim 151, selected from the group consisting of symptom, frontotemporal dementia, type II diabetes, systemic amyloidosis, serum amyloidosis, British familial dementia, Danish familial dementia, macular degeneration, and cerebrovascular amyloidosis. 152. The method of claim 151, wherein the disease is Alzheimer's disease. The composition is intrathecal, intrathecal, oral, transdermal, pulmonary, intravenous, subcutaneous, nasal, intraarterial, intracranial, intradermal, intraperitoneal 143. The method of claim 142, wherein the method is administered by a method selected from the group consisting of: intramuscular administration, rectal administration, and buccal administration. A method of making an antibody comprising:
A. A method comprising obtaining a conformational epitope of a fibrillar aggregate that forms in a human or animal and contributes to amyloid fibril formation. 165. The method of claim 155, wherein step A comprises recovering the antibody from a human or animal. A method of making an antibody comprising:
A. Administering to a human or animal a composition comprising an epitope of a fibrillar aggregate that forms in a human or animal and contributes to amyloid fibril formation;
The method wherein the amyloid fibril is substantially free of the epitope. 158. The method of claim 157, wherein step A comprises recovering the antibody from the human or animal. A method of diagnosing a disease characterized by amyloid deposits, the method comprising:
A. Mixing a tissue or fluid from a human or animal patient with a composition comprising an antibody that binds to a conformational epitope of a fibrillar aggregate that forms in a human or animal and contributes to amyloid fibril formation. Including methods. 160. The method of claim 159, wherein the disease is Alzheimer's disease. 160. The method of claim 159, wherein the tissue or fluid is cerebrospinal fluid. A method of diagnosing a disease characterized by amyloid deposits, the method comprising:
A. Mixing a tissue or fluid from a human or animal patient with a composition comprising an antibody that binds to an epitope of a fibrillar aggregate that forms in a human or animal and contributes to amyloid fibril formation;
The method wherein the amyloid fibril is substantially free of the epitope. 163. The method of claim 162, wherein the disease is Alzheimer's disease. 163. The method of claim 162, wherein the tissue or fluid is cerebrospinal fluid. A method for assessing the effectiveness of a method of treating a human or animal having a disease characterized by amyloid deposition comprising:
A. Prior to treatment with a drug, the baseline amount of antibodies specific to conformational epitopes of primordial fibrillar aggregates that form in humans or animals and contribute to amyloid fibril formation in patient-derived tissue samples Determining, and B. Comparing the amount of the antibody in the tissue sample from the subject after treatment with the agent to a baseline amount of the antibody. 166. The method of claim 165, wherein a decrease in the amount of the antibody measured after the treatment and compared to a baseline amount of the antibody or a lack of significant difference therebetween indicates a negative treatment result. 166. The method of claim 165, wherein a significantly greater amount of the antibody measured after the treatment and compared to a baseline amount of the antibody indicates a positive treatment outcome. 166. The method of claim 165, wherein the amount of antibody is measured as antibody titer. 166. The method of claim 165, wherein the amount of antibody is measured by an ELISA assay. A method for assessing the efficacy of a method of treating a human or animal having a disease characterized by amyloid deposition, the method comprising:
A. Prior to treatment with a drug, determining a baseline amount in a patient-derived tissue sample of an antibody specific for an epitope of a fibrillar aggregate that forms in a human or animal and contributes to amyloid fibril formation The amyloid fibril is substantially free of the epitope,
B. Comparing the amount of the antibody in the tissue sample from the subject after treatment with the agent to a baseline amount of the antibody. 171. The method of claim 170, wherein a decrease in the amount of the antibody measured after the treatment and compared to a baseline amount of the antibody or a lack of significant difference therebetween indicates a negative treatment result. 171. The method of claim 170, wherein a significantly greater amount of the antibody measured after the treatment and compared to a baseline amount of the antibody indicates a positive treatment outcome. 171. The method of claim 170, wherein the amount of antibody is measured as antibody titer. 171. The method of claim 170, wherein the amount of antibody is measured by an ELISA assay. A diagnostic kit useful for detecting diseases characterized by amyloid deposits, which binds to conformational epitopes of primordial fibril aggregates that form in humans or animals and contribute to amyloid fibril formation A kit comprising a composition containing the antibody to be treated. A diagnostic kit useful for detecting a disease characterized by amyloid deposition, the kit comprising an antibody that binds to an epitope of a protofibrillar aggregate that forms in a human or animal and contributes to amyloid fibril formation. A kit comprising an isolated composition comprising, wherein the amyloid fibril is substantially free of the epitope. A method for assessing the ability of a test substance to inhibit the formation of an amyloid oligomeric intermediate comprising:
A. Preparing a first mixture comprising the test substance and amyloid peptide;
B. Incubating the first mixture in the absence of any inhibitory effect and under conditions where an amyloid oligomeric intermediate is formed from the amyloid peptide;
C. C. preparing a second mixture by mixing the incubated mixture from step B with an antibody that recognizes the conformational epitope of the oligomeric intermediate; Determining the amount of oligomeric intermediate formed based on the binding of the antibody to the conformational epitope of the oligomeric intermediate. 178. The method of claim 177, wherein step D is performed by ELISA. A method for assessing the ability of a test substance to cause degradation, dissociation or substantial destruction of an amyloid oligomer intermediate comprising:
A. Preparing a first mixture comprising an amyloid oligomer intermediate and the test substance;
B. C. preparing a second mixture by mixing the mixture from step A with an antibody that recognizes a conformational epitope of the oligomeric intermediate; Determining the amount of amyloid oligomer intermediate that has not undergone degradation, dissociation or substantial destruction based on binding of the antibody to the conformational epitope of the oligomer intermediate remaining in the second mixture. Including methods. 178. The method of claim 178, wherein step C is performed by ELISA.

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